**3.2 BRCA1 structure modifications found in cancer**

Mutations in brca1 and brca2 genes have been found in 30-50% of hereditary breast and ovarian cancers. Women carrying BRCA1 mutations are particularly susceptible to the development of breast or ovarian cancer at an age earlier than 35-40 years old with a probability rate of 45-60% and 20-40%, respectively.

Most cancer-associated BRCA1 mutations identified so far, result in the premature translational termination of the protein and influence BRCA1 integrity and function. A large number of missense mutations is located in BRCT tandem repeats of BRCA1, while only few of them may cause loss of the protein's function, abolition of protein interactions and protein miss-localization. Therefore, it seems that the BRCT repeats in BRCA1 are essential for the tumor suppressing function of the protein as protein truncation and missense variants within the BRCT domain have been shown to be associated with human breast and ovarian cancers.

Variants that result in large truncations are deleterious to function and therefore can be classified as disease-associated. In contrast, missense mutations typically remain unclassified. Thus, the BIC database currently contains more than 108 missense mutations in the BRCT domains of BRCA1, but only 7% of them have been classified. These missense mutations may be either polymorphisms or mutations predisposing the carrier to cancer progression. The variants D1692Y, C1697R, R1699W, A1708E, S1715R, P1749R and M1775R all appear to be associated with an increased risk of breast cancer, while M1625I appears to be a benign polymorphism (Williams et al., 2003). Unfortunately, most of the missense mutations could not been assessed for disease association. An attempt to classify these variants by measuring the thermodynamic stability of the BRCA1 BRCT domains resulted in investigation of the effects of 36 missense mutations (Rowling et al., 2010). The mutations show a range of effects. Some do not change the stability, whereas others destabilize the protein by as much as 6 kcal mol−1; one-third of the mutants were considered to destabilize the protein by an even greater amount, as they could not be expressed in soluble form in *Escherichia coli*. Several computer algorithms were used in an attempt to predict the mutant effects. According to these results the variants were grouped into two classes (destabilizing by less than or more than 2.2 kcal mol−1). Importantly, with the exception of the few mutants located in the binding site, none showed a significant reduction in affinity for phosphorylated substrate. These results indicate

Structure-Function Relationship of DNA Repair

Proteins: Lessons from BRCA1 and RAD51 Studies 113

the BRCT domain altered BRCA1 localization, causing the protein to be excluded from the nucleus. Two of the C-terminal mutations (M1775R and Y1853X) that restricted nuclear localization are identical to mutations that disrupt BRCA1 C-terminal folding, suggesting that the conformational changes they elicit might be deleterious to BRCA1 nuclear transport. This nuclear exclusion was not due to increased nuclear export, but to reduced nuclear import. Similar findings were observed for both the overexpressed and endogenous forms of the BRCT mutant, BRCA1 (5382insC). Also, Chen et al., 1995 have published controversial findings, which claimed that BRCA1 was detected almost exclusively in the cytoplasm in breast cancer

In our laboratory more than fifteen BRCA1-BRCT proteins mutants have already been studied for structural and functional alterations in protein's integrity. The most destabilizing protein mutants such as M1775K, V1809F (Fig3) were collected in order to be examined in cellular level about their impact in BRCA1 subcellular compartmentalization. M1775K is a rare breast cancer-linked mutation and it has been identified only in two unrelated families of European ancestry with a history of breast cancer. Met1775 is strongly involved in the phosphopeptidebinding pocket of the BRCT domain. The mutation of Met1775, namely the mutation M1775R, is much more frequent worldwide among patients with hereditary breast/ovarian cancer, its association with the disease is epidemiologically established and was the first characterized to be linked to cancer. The M1775R mutation has already shown to change the intracellular localization of BRCA1 protein which is less focused into the nucleus. The M1775K missense variant according to our *in vitro* experiments fails to bind to synthetic peptides such pBACH1/BRIP1 or pCtIP. Structural analysis of the interatomic interactions of Lys1775 show a direct clash of its side chain with Phe 13 of either phosphopeptide, a result arising from the

Fig. 3. DSC profiles for the thermally induced denaturation of BRCT-wt and of five missense

V1809F is a rare mutation linked to hereditary breast/ ovarian cancer. Only a few cases of the mutation have been submitted to the BIC database with loss of function reported by *in vitro* experiments, regarding the interactions with synthetic phosphopeptides pBACH1/BRIP1 and pCtIP. The residue Val1809 is conserved among species. Val1809 and Met1775 are crucial for the integrity of phosphopeptide binding pocket of the BRCA1 protein and exhibit no binding to either pBACH1/BRIP1 or pCtIP synthetic phosphopeptides. These results, in combination with the fact that V1809F resembles structural destabilization of the native fold similar to M1775R, strongly supports the

variants V1809F, M1783T, M1775K, P1812A, and V1696L. (from Drikos et al., 2009).

tissues, but remained nuclear in normal tissue and in other cancer cell types.

disruption of the BRCT-phosphopeptide binding pocket.

classification of V1809F as pathogenic.

that despite very large losses in stability, the integrity of the structure is not compromised by the mutations. Thus, the majority of mutations seem to cause loss of function by reducing the proportion of BRCA1 molecules that are in the folded state and increasing the proportion of molecules that are unfolded. The authors predict that small molecule stabilization of the structure could be a generally applicable preventative therapeutic strategy for rescuing many BRCA1 mutations. Another recent approach by Lee et al., 2010, extended in 117 variants, comprehensively shows how functional and structural information can be useful in the development of models to assess cancer risk.

Cancer-associated mutations in the BRCT domain of BRCA1 (BRCA1-BRCT) abolish its tumor suppressor function by disrupting interactions with other proteins such as BACH1. Many cancer-related mutations do not cause sufficient destabilization to lead to global unfolding under physiological conditions, and thus abrogation of function probably is due to localized structural changes. Molecular dynamics simulations on three cancer-associated mutants, A1708E, M1775R, and Y1853ter, and on the wild type and benign M1652I mutant, followed by comparison of the structures and fluctuations showed that only the cancerassociated mutants exhibited significant backbone structure differences from the wild-type crystal structure in BACH1-binding regions, some of which are far from the mutation sites. These BACH1-binding regions of the cancer-associated mutants also exhibited increases in their fluctuation magnitudes compared with the same regions in the wild type and M1562I mutant, as quantified by quasiharmonic analysis. The increased fluctuations in the diseaserelated mutants suggest an increase in vibrational entropy in the unliganded state that could result in a larger entropy loss in the disease-related mutants upon binding BACH1 than in the wild type. Vibrational entropies of the A1708E and wild type in the free state and bound to a BACH1-derived phosphopeptide, calculated using quasiharmonic analysis, determined the binding entropy difference DeltaDeltaS between the A1708E mutant and the wild type. In overall such biophysical/biochemical studies supported by suited algorithms showed that the observed differences in structure, flexibility, and entropy of binding are likely to be responsible for abolition of BACH1 binding, and illustrate that many disease-related mutations could have very long-range effects. Such methods have potential for identifying correlated motions responsible for other long-range effects of deleterious mutations. (Gough et al., 2007)

The C-terminal BRCT domains are also evidenced to mediate the transcriptional activity of BRCA1. Most of the published mutations within the BRCT domains have been reported to affect BRCA1 nuclear functions including DNA repair and transcriptional activity. The biochemical and biophysical studies of our group have already demonstrated that mutations of the BRCT domain: (i) affected the folding of the domain to a varying degree depending on the induced destabilization and (ii) altered and abolished the affinity of BRCT domain to synthetic phosphopeptides corresponding to BRCT interacting regions of pBACH1/ BRIP1 and pCtIP, by affecting the structural integrity of the BRCT active sites.

BRCA1 is a nuclear-cytoplasmic shuttling protein and its nuclear localization is regulated by the combined action of nuclear localization (NLS) and nuclear export signals (NES). In most cases, however, cellular and ectopically expressed BRCA1 are primarily nuclear due to nuclear import mediated by the two NLSs and interaction with the RING domain binding protein, BARD1, which can carry BRCA1 into the nucleus and trap it there by masking its nuclear export signal.

Despite the structural studies of BRCA1-BRCT protein mutants, the influence of these mutations at protein localization in cellular level has not yet been adequately addressed. Only few of them have already determined to present protein mislocalization. BRCA1 mutations of

that despite very large losses in stability, the integrity of the structure is not compromised by the mutations. Thus, the majority of mutations seem to cause loss of function by reducing the proportion of BRCA1 molecules that are in the folded state and increasing the proportion of molecules that are unfolded. The authors predict that small molecule stabilization of the structure could be a generally applicable preventative therapeutic strategy for rescuing many BRCA1 mutations. Another recent approach by Lee et al., 2010, extended in 117 variants, comprehensively shows how functional and structural information can be useful in the

Cancer-associated mutations in the BRCT domain of BRCA1 (BRCA1-BRCT) abolish its tumor suppressor function by disrupting interactions with other proteins such as BACH1. Many cancer-related mutations do not cause sufficient destabilization to lead to global unfolding under physiological conditions, and thus abrogation of function probably is due to localized structural changes. Molecular dynamics simulations on three cancer-associated mutants, A1708E, M1775R, and Y1853ter, and on the wild type and benign M1652I mutant, followed by comparison of the structures and fluctuations showed that only the cancerassociated mutants exhibited significant backbone structure differences from the wild-type crystal structure in BACH1-binding regions, some of which are far from the mutation sites. These BACH1-binding regions of the cancer-associated mutants also exhibited increases in their fluctuation magnitudes compared with the same regions in the wild type and M1562I mutant, as quantified by quasiharmonic analysis. The increased fluctuations in the diseaserelated mutants suggest an increase in vibrational entropy in the unliganded state that could result in a larger entropy loss in the disease-related mutants upon binding BACH1 than in the wild type. Vibrational entropies of the A1708E and wild type in the free state and bound to a BACH1-derived phosphopeptide, calculated using quasiharmonic analysis, determined the binding entropy difference DeltaDeltaS between the A1708E mutant and the wild type. In overall such biophysical/biochemical studies supported by suited algorithms showed that the observed differences in structure, flexibility, and entropy of binding are likely to be responsible for abolition of BACH1 binding, and illustrate that many disease-related mutations could have very long-range effects. Such methods have potential for identifying correlated motions responsible for other long-range effects of deleterious mutations.

The C-terminal BRCT domains are also evidenced to mediate the transcriptional activity of BRCA1. Most of the published mutations within the BRCT domains have been reported to affect BRCA1 nuclear functions including DNA repair and transcriptional activity. The biochemical and biophysical studies of our group have already demonstrated that mutations of the BRCT domain: (i) affected the folding of the domain to a varying degree depending on the induced destabilization and (ii) altered and abolished the affinity of BRCT domain to synthetic phosphopeptides corresponding to BRCT interacting regions of pBACH1/ BRIP1

BRCA1 is a nuclear-cytoplasmic shuttling protein and its nuclear localization is regulated by the combined action of nuclear localization (NLS) and nuclear export signals (NES). In most cases, however, cellular and ectopically expressed BRCA1 are primarily nuclear due to nuclear import mediated by the two NLSs and interaction with the RING domain binding protein, BARD1, which can carry BRCA1 into the nucleus and trap it there by masking its

Despite the structural studies of BRCA1-BRCT protein mutants, the influence of these mutations at protein localization in cellular level has not yet been adequately addressed. Only few of them have already determined to present protein mislocalization. BRCA1 mutations of

and pCtIP, by affecting the structural integrity of the BRCT active sites.

development of models to assess cancer risk.

(Gough et al., 2007)

nuclear export signal.

the BRCT domain altered BRCA1 localization, causing the protein to be excluded from the nucleus. Two of the C-terminal mutations (M1775R and Y1853X) that restricted nuclear localization are identical to mutations that disrupt BRCA1 C-terminal folding, suggesting that the conformational changes they elicit might be deleterious to BRCA1 nuclear transport. This nuclear exclusion was not due to increased nuclear export, but to reduced nuclear import. Similar findings were observed for both the overexpressed and endogenous forms of the BRCT mutant, BRCA1 (5382insC). Also, Chen et al., 1995 have published controversial findings, which claimed that BRCA1 was detected almost exclusively in the cytoplasm in breast cancer tissues, but remained nuclear in normal tissue and in other cancer cell types.

In our laboratory more than fifteen BRCA1-BRCT proteins mutants have already been studied for structural and functional alterations in protein's integrity. The most destabilizing protein mutants such as M1775K, V1809F (Fig3) were collected in order to be examined in cellular level about their impact in BRCA1 subcellular compartmentalization. M1775K is a rare breast cancer-linked mutation and it has been identified only in two unrelated families of European ancestry with a history of breast cancer. Met1775 is strongly involved in the phosphopeptidebinding pocket of the BRCT domain. The mutation of Met1775, namely the mutation M1775R, is much more frequent worldwide among patients with hereditary breast/ovarian cancer, its association with the disease is epidemiologically established and was the first characterized to be linked to cancer. The M1775R mutation has already shown to change the intracellular localization of BRCA1 protein which is less focused into the nucleus. The M1775K missense variant according to our *in vitro* experiments fails to bind to synthetic peptides such pBACH1/BRIP1 or pCtIP. Structural analysis of the interatomic interactions of Lys1775 show a direct clash of its side chain with Phe 13 of either phosphopeptide, a result arising from the disruption of the BRCT-phosphopeptide binding pocket.

Fig. 3. DSC profiles for the thermally induced denaturation of BRCT-wt and of five missense variants V1809F, M1783T, M1775K, P1812A, and V1696L. (from Drikos et al., 2009).

V1809F is a rare mutation linked to hereditary breast/ ovarian cancer. Only a few cases of the mutation have been submitted to the BIC database with loss of function reported by *in vitro* experiments, regarding the interactions with synthetic phosphopeptides pBACH1/BRIP1 and pCtIP. The residue Val1809 is conserved among species. Val1809 and Met1775 are crucial for the integrity of phosphopeptide binding pocket of the BRCA1 protein and exhibit no binding to either pBACH1/BRIP1 or pCtIP synthetic phosphopeptides. These results, in combination with the fact that V1809F resembles structural destabilization of the native fold similar to M1775R, strongly supports the classification of V1809F as pathogenic.

Structure-Function Relationship of DNA Repair

**V1809F**

**wt** 

their classification regarding to cancer-relation.

**3.3 The RAD51 structure – function relation paradigm** 

cytoplasm.

Proteins: Lessons from BRCA1 and RAD51 Studies 115

**M1652I**

**M1775K**

Fig. 4. Subcellular mis-localization of cancer linked GFP-BRCA1-BRCT mutations, M1775K and V1809F in the cytoplasm of MCF-7 cells in contrast to the wild type BRCA1 (wt) and the

A living cell is a dynamic unit with flexible equilibrium between different processes which drive cell fate and determination decisions. The various pathways involved are either activated or suppressed as a result of qualitative and /or quantitative interactions between biomolecules. BRCA1 is an elegant paradigm of both kinds of interactions. Truncated or absent BRCA1 (abolishment of qualitative interactions) leads to impaired DNA repair, carcinogenesis and cancer progression. As indicated by the studies of various missense mutations there are cases where although the mutated BRCA1 seems to function properly, a significant proportion of BRCA1 molecules adopts an unfolding state and only few molecules are found in a given time in proper and functional structure. M1324K, R42573L mutations are good paradigms where biophysical studies of these BRCA1 mutants indicated that although interactions with phosphopeptides were attained, the majority of mutant molecules were detected in improper folding state. Moreover, as many missense variants remain to be characterized, combination of biophysical with cell/molecular biology studies, as in the case of M1775K and V1809F mutations, is expected to substantially contribute in

DNA double strand breaks (DSBs), produced by either exogenous causes or in order to restore stalled replication forks during genome replication, are detrimental to cell survival and are mainly restored by either Homologous Recombination Repair (HRR) or Non-Homologous End Joining (NHEJ). The two pathways compete for each other while a number of factors such as cell cycle stage and availability of duplicated DNA regulate the

'neutral' mutation of BRCT, M1652I which are detected in both the nucleus and the

Additionally, the variant M1652I is located at the first tandem of the BRCT domain and seems to have neutral influence on breast cancer pathogenesis. Based on preliminary structural studies by our laboratory, this variant is less involved in structural alteration of BRCT but further analysis is required. M1652I is classified as low risk mutation. Therefore we decided to include it to our study in order to compare it with more destabilizing mutants such as V1809F and M1775K.

In order to assess how the selected BRCA1-BRCT mutants influence the subcellular localization of BRCA1, we produced BRCA1-GFP fusion proteins with the corresponding mutations introduced at the BRCT domain. The GFP-BRCA1-BRCT mutated proteins were inserted into MCF-7 cells and their subcellular localization was assessed by fluorescent microscopy.

According to our results, destabilizing mutations of the carboxyl terminal region of BRCA1 seem to influence protein localization and presumably DDR. As shown in Fig 4, BRCA1- V1809F-GFP and M17775K are restricted to the cytoplasm in contrast to the nuclearcytoplasmic localization of BRCA1wt and M1652I. As EGFP-BRCA1-M1652I shows similar subcellular distribution to the BRCA1wt–GFP protein (detected both in the nucleus and the cytoplasm), it is presumed that the structural change caused by replacement of M1652 to Ile has a minor effect of BRCA1 nuclear transport. UV irradiation of cells expressing wt or the mutants mentioned failed to drive M1175K and V1809F to the cell nucleus in contrast to both wt and M1652I which were then detected exclusively in the nucleus and shown to at least in part colocalize with Rad51 foci (data not shown from Drikos et al., submitted). Mutations such as M1775K and V1809F which disrupt BRCA1 C-terminal folding, appears that result to conformational and functional changes which might be restrictive to BRCA1 nuclear transport in contrast to more mild missense variants such as M1652I. These results suggest that structural integrity modifications of the BRCA1-BRCT domain can be reflected to the protein's subcellular localization and therefore can serve for further characterization and classification of the variant, in combination with the structural data (table 1). M1175K and V1809F are located near to the binding site of the inter-repeat region and affect through hydrophobic interactions the structural and functional integrity of the domain.


Table 1. Summary of the impact of BRCA1-BRCT mutants on the structural, functional and cellular levels. Mutations such as M1775K and V1809F, which disrupt BRCA1 C-terminal folding, induce also alterations of the integrity of the BRCA1-BRCT domain and the proteins subcellular localization.

Additionally, the variant M1652I is located at the first tandem of the BRCT domain and seems to have neutral influence on breast cancer pathogenesis. Based on preliminary structural studies by our laboratory, this variant is less involved in structural alteration of BRCT but further analysis is required. M1652I is classified as low risk mutation. Therefore we decided to include it to our study in order to compare it with more destabilizing mutants

In order to assess how the selected BRCA1-BRCT mutants influence the subcellular localization of BRCA1, we produced BRCA1-GFP fusion proteins with the corresponding mutations introduced at the BRCT domain. The GFP-BRCA1-BRCT mutated proteins were inserted into MCF-7 cells and their subcellular localization was assessed by fluorescent

According to our results, destabilizing mutations of the carboxyl terminal region of BRCA1 seem to influence protein localization and presumably DDR. As shown in Fig 4, BRCA1- V1809F-GFP and M17775K are restricted to the cytoplasm in contrast to the nuclearcytoplasmic localization of BRCA1wt and M1652I. As EGFP-BRCA1-M1652I shows similar subcellular distribution to the BRCA1wt–GFP protein (detected both in the nucleus and the cytoplasm), it is presumed that the structural change caused by replacement of M1652 to Ile has a minor effect of BRCA1 nuclear transport. UV irradiation of cells expressing wt or the mutants mentioned failed to drive M1175K and V1809F to the cell nucleus in contrast to both wt and M1652I which were then detected exclusively in the nucleus and shown to at least in part colocalize with Rad51 foci (data not shown from Drikos et al., submitted). Mutations such as M1775K and V1809F which disrupt BRCA1 C-terminal folding, appears that result to conformational and functional changes which might be restrictive to BRCA1 nuclear transport in contrast to more mild missense variants such as M1652I. These results suggest that structural integrity modifications of the BRCA1-BRCT domain can be reflected to the protein's subcellular localization and therefore can serve for further characterization and classification of the variant, in combination with the structural data (table 1). M1175K and V1809F are located near to the binding site of the inter-repeat region and affect through

hydrophobic interactions the structural and functional integrity of the domain.

hydrophobic Deleterious Destabilizing Alter binding

hydrophobic Deleterious Destabilizing Alter binding

**BRCA1wt** Nuclear-

**Structural Stability** 

No effect Neutral No effect Unknown Nuclear-

Table 1. Summary of the impact of BRCA1-BRCT mutants on the structural, functional and cellular levels. Mutations such as M1775K and V1809F, which disrupt BRCA1 C-terminal folding, induce also alterations of the integrity of the BRCA1-BRCT domain and the proteins

**Functional Activity with synthetic peptides** 

**Subcellular localization (-UV)** 

affinity Cytoplasmic Cytoplasmic

affinity Cytoplasmic Cytoplasmic

Cytoplasmic Nuclear

Cytoplasmic Nuclear

**Subcellular localization (+UV)** 

**Cancer Risk of the mutation** 

such as V1809F and M1775K.

**Effect of mutation** 

Potential

Potential

microscopy.

**GFP-BRCA1- V1809F** 

**GFP-BRCA1- M1775K** 

**GFP-BRCA1- M1652I** 

**GFP-**

subcellular localization.

Fig. 4. Subcellular mis-localization of cancer linked GFP-BRCA1-BRCT mutations, M1775K and V1809F in the cytoplasm of MCF-7 cells in contrast to the wild type BRCA1 (wt) and the 'neutral' mutation of BRCT, M1652I which are detected in both the nucleus and the cytoplasm.

A living cell is a dynamic unit with flexible equilibrium between different processes which drive cell fate and determination decisions. The various pathways involved are either activated or suppressed as a result of qualitative and /or quantitative interactions between biomolecules. BRCA1 is an elegant paradigm of both kinds of interactions. Truncated or absent BRCA1 (abolishment of qualitative interactions) leads to impaired DNA repair, carcinogenesis and cancer progression. As indicated by the studies of various missense mutations there are cases where although the mutated BRCA1 seems to function properly, a significant proportion of BRCA1 molecules adopts an unfolding state and only few molecules are found in a given time in proper and functional structure. M1324K, R42573L mutations are good paradigms where biophysical studies of these BRCA1 mutants indicated that although interactions with phosphopeptides were attained, the majority of mutant molecules were detected in improper folding state. Moreover, as many missense variants remain to be characterized, combination of biophysical with cell/molecular biology studies, as in the case of M1775K and V1809F mutations, is expected to substantially contribute in their classification regarding to cancer-relation.

#### **3.3 The RAD51 structure – function relation paradigm**

DNA double strand breaks (DSBs), produced by either exogenous causes or in order to restore stalled replication forks during genome replication, are detrimental to cell survival and are mainly restored by either Homologous Recombination Repair (HRR) or Non-Homologous End Joining (NHEJ). The two pathways compete for each other while a number of factors such as cell cycle stage and availability of duplicated DNA regulate the

Structure-Function Relationship of DNA Repair

Lazao-Trueba & Silva, 2006).

Proteins: Lessons from BRCA1 and RAD51 Studies 117

cell cycle control and apoptosis (Henning & Sturzbecher, 2003; Gatz & Wiesmuller, 2006;

Scheme 3. Simplified description of HRR major steps. The presynaptic phase involves resection of the 5' terminated strand at the DNA double strand break point (black line) and the formation of the RAD51 active nucleoprotein filament on 3'ssDNA tails. During the synaptic phase RAD51 traces the homologous strand (grey line)(usually the sister chromatid) and performs the strand exchange. After DNA heteroduplex extension and branch migration (newly synthesized DNA is shown as framed grey line) the Holiday junctions produced are separated resulting in two intact homologous DNA molecules. (Resolution or dissolution of holiday junction may also involve crossing over resulting in

RAD51 function is mainly controlled by the breast cancer susceptibility gene 2 product BRCA2 which acts as a recombination mediator (scheme 4). Briefly, BRCA2 targets RAD51 to ssDNA for assembly into a nucleoprotein filament, stabilizes the ATP-bound form of RAD51 and inhibits RAD51 assembly on dsDNA (Shivji et al., 2009). BRCA2 is an extremely large protein of 3418 residues and essentially contributes to RAD51-mediated HRR through several regions. RAD51 interacts with 8 copies of ~35 residues repeated motifs (BRC repeats) located at exon 11 (Yu et al., 2003), as well as with an unrelated carboxyl-terminal motif in exon 27 (Esashi et al., 2007). The BRC repeats sequence, unlike the C-terminal motif, is

chimeric but still homologous DNA molecules).

final choice. HRR is the prominent (high fidelity) DSB repair pathway, requiring an homologous DNA sequence present (the identical sister chromatid located in proper distance) and therefore is mainly active during S/G2 phases of cell cycle, while the error prone NHEJ pathway is mainly active during G1 and early S phase, although in certain cases can work in all cell cycle phases. HRR compete NHEJ pathway through a complicated manner, where initial DNA damage signalling factors play important roles. Damage processing and effectiveness of repair are incessantly checked by sensor molecules which through a series of distinct but interconnected pathways prolong cell cycle arrest, induce senescence or apoptosis depending on the information flow and the signals produced (Freeman & Monteiro 2010).

An important early step in HRR is the conversion of double to single stranded DNA in the area of the double strand break, which in turn is coated by the Replication Protein A (RPA) and can be extended up to 4 – 5 Kb on both sides along the break point. Displacement of RPA by RAD51, assembled as a nucleoprotein filament on the ssDNA, is the initial step towards HRR and is highly regulated through interactions with a variety of accessory proteins referred to as the 'recombination mediators' (Essers et al., 2002; Henning & Sturzbecher 2003; Schild & Wiese, 2010; Forget & Kowalczykowski, 2010; West, 2003; San Filippo et al., 2008; Li & Heyer, 2008). The central event in HRR is the synapsis of the singlestranded (ss)DNA molecule - produced along the double strand break point - with homologous duplex DNA. The strand invasion is mediated by the strand transferase RAD51 oligomerized on ssDNA as an active nucleoprotein filament (and the corresponding cofactors needed for filament assembly and function), which initiates the strand exchange that leads to recombination. RAD51, a recombinase essential for cell viability, is one of the most conserved molecules known. RAD51 mediates strand exchange via distinct reactions grouped into the presynaptic, synaptic, and postsynaptic phases (2) (Heyer, 2007; Shivji et al., 2009). The major steps of HRR process are schematically illustrated in Scheme 3.

RAD51 assembly on ssDNA and subsequent catalysis of homology dependent strand invasion is mainly driven by the tumor suppressor protein BRCA2 while during the different phases of HRR RAD51 interacts sequentially with other molecules involved in HRR, cell cycle control and cell fate decisions. RAD51, as part of dynamic structures called DNA damage foci, seems to be a stably associated core component, whereas other co-factors such as Rad52 and Rad54 rapidly and reversibly interact with the structure (Essers et al., 2002). RAD51 function depends on protein re-localization and is mainly regulated by various post-translational modifications, mainly phosphorylation (Slupianek et al., 2001; Venkitaraman, 2001), as well as non-covalent interaction with SUMO (Ouyang et al., 2009). Along evolution recombinase molecules are highly conserved, starting from the prokaryotic orthologue RecA to mammalian RAD51. The fact the RecA seems not to be an ancestor of RAD51 but these two molecules are considered to have evolved by converging evolution, suggests that the structure obtained is crucial for the specific recombination function and cannot afford modifications. This notion is further supported by the absence of RAD51 coding region mutants in any cancer type, while cells or animals that do not express RAD51 eventually are not viable (Tsuzuki et al., 1996; Sonoda et al., 1998). Despite the non-detection of RAD51 mutants itself it is clear that mutations in recombination mediators and comediators, which control RAD51 activity and availability, are highly related to cancer susceptibility and particularly breast cancer (Venkitaraman, 2009; Rahan et al., 2007; Seviour & Lin, 2010). Additionally, the 'guardian of the genome' p53, found mutated in more than 50% of cancers, also directly interacts with RAD51 presumably connecting HRR efficiency to

final choice. HRR is the prominent (high fidelity) DSB repair pathway, requiring an homologous DNA sequence present (the identical sister chromatid located in proper distance) and therefore is mainly active during S/G2 phases of cell cycle, while the error prone NHEJ pathway is mainly active during G1 and early S phase, although in certain cases can work in all cell cycle phases. HRR compete NHEJ pathway through a complicated manner, where initial DNA damage signalling factors play important roles. Damage processing and effectiveness of repair are incessantly checked by sensor molecules which through a series of distinct but interconnected pathways prolong cell cycle arrest, induce senescence or apoptosis depending on the information flow and the signals produced

An important early step in HRR is the conversion of double to single stranded DNA in the area of the double strand break, which in turn is coated by the Replication Protein A (RPA) and can be extended up to 4 – 5 Kb on both sides along the break point. Displacement of RPA by RAD51, assembled as a nucleoprotein filament on the ssDNA, is the initial step towards HRR and is highly regulated through interactions with a variety of accessory proteins referred to as the 'recombination mediators' (Essers et al., 2002; Henning & Sturzbecher 2003; Schild & Wiese, 2010; Forget & Kowalczykowski, 2010; West, 2003; San Filippo et al., 2008; Li & Heyer, 2008). The central event in HRR is the synapsis of the singlestranded (ss)DNA molecule - produced along the double strand break point - with homologous duplex DNA. The strand invasion is mediated by the strand transferase RAD51 oligomerized on ssDNA as an active nucleoprotein filament (and the corresponding cofactors needed for filament assembly and function), which initiates the strand exchange that leads to recombination. RAD51, a recombinase essential for cell viability, is one of the most conserved molecules known. RAD51 mediates strand exchange via distinct reactions grouped into the presynaptic, synaptic, and postsynaptic phases (2) (Heyer, 2007; Shivji et

al., 2009). The major steps of HRR process are schematically illustrated in Scheme 3.

RAD51 assembly on ssDNA and subsequent catalysis of homology dependent strand invasion is mainly driven by the tumor suppressor protein BRCA2 while during the different phases of HRR RAD51 interacts sequentially with other molecules involved in HRR, cell cycle control and cell fate decisions. RAD51, as part of dynamic structures called DNA damage foci, seems to be a stably associated core component, whereas other co-factors such as Rad52 and Rad54 rapidly and reversibly interact with the structure (Essers et al., 2002). RAD51 function depends on protein re-localization and is mainly regulated by various post-translational modifications, mainly phosphorylation (Slupianek et al., 2001; Venkitaraman, 2001), as well as non-covalent interaction with SUMO (Ouyang et al., 2009). Along evolution recombinase molecules are highly conserved, starting from the prokaryotic orthologue RecA to mammalian RAD51. The fact the RecA seems not to be an ancestor of RAD51 but these two molecules are considered to have evolved by converging evolution, suggests that the structure obtained is crucial for the specific recombination function and cannot afford modifications. This notion is further supported by the absence of RAD51 coding region mutants in any cancer type, while cells or animals that do not express RAD51 eventually are not viable (Tsuzuki et al., 1996; Sonoda et al., 1998). Despite the non-detection of RAD51 mutants itself it is clear that mutations in recombination mediators and comediators, which control RAD51 activity and availability, are highly related to cancer susceptibility and particularly breast cancer (Venkitaraman, 2009; Rahan et al., 2007; Seviour & Lin, 2010). Additionally, the 'guardian of the genome' p53, found mutated in more than 50% of cancers, also directly interacts with RAD51 presumably connecting HRR efficiency to

(Freeman & Monteiro 2010).

cell cycle control and apoptosis (Henning & Sturzbecher, 2003; Gatz & Wiesmuller, 2006; Lazao-Trueba & Silva, 2006).

Scheme 3. Simplified description of HRR major steps. The presynaptic phase involves resection of the 5' terminated strand at the DNA double strand break point (black line) and the formation of the RAD51 active nucleoprotein filament on 3'ssDNA tails. During the synaptic phase RAD51 traces the homologous strand (grey line)(usually the sister chromatid) and performs the strand exchange. After DNA heteroduplex extension and branch migration (newly synthesized DNA is shown as framed grey line) the Holiday junctions produced are separated resulting in two intact homologous DNA molecules. (Resolution or dissolution of holiday junction may also involve crossing over resulting in chimeric but still homologous DNA molecules).

RAD51 function is mainly controlled by the breast cancer susceptibility gene 2 product BRCA2 which acts as a recombination mediator (scheme 4). Briefly, BRCA2 targets RAD51 to ssDNA for assembly into a nucleoprotein filament, stabilizes the ATP-bound form of RAD51 and inhibits RAD51 assembly on dsDNA (Shivji et al., 2009). BRCA2 is an extremely large protein of 3418 residues and essentially contributes to RAD51-mediated HRR through several regions. RAD51 interacts with 8 copies of ~35 residues repeated motifs (BRC repeats) located at exon 11 (Yu et al., 2003), as well as with an unrelated carboxyl-terminal motif in exon 27 (Esashi et al., 2007). The BRC repeats sequence, unlike the C-terminal motif, is

Structure-Function Relationship of DNA Repair

genomic stability (Petalcorin et al., 2007).

Proteins: Lessons from BRCA1 and RAD51 Studies 119

RAD51 filaments are further stabilized by direct interaction of the BRCA2 C terminus to the interface created by two adjacent RAD51 protomers. This way filaments cannot be dissociated by the BRC repeats. Interaction of the BRCA2 C terminus with the RAD51 filament causes a large movement of the flexible RAD51 N-terminal domain that is important in regulating filament dynamics. RAD51 interaction with the BRCA2 C-terminal region may facilitate efficient nucleation of RAD51 multimers on DNA and thereby stimulate recombination-mediated repair. (Esashi et al., 2007). Data from studies the Caenorhabditis elegans BRCA2 homolog CeBRC-2 support a model where an interaction with RAD-51 alone is likely involved in filament nucleation, whereas a second independent interaction is involved in *in situ* stabilization of RAD51 filaments by BRCA2 and provide further insight into why mutations in many different positions within BRCA2 lead to loss of

RAD51, the central homology strand search and strand exchange effector in HRR can serve as a nice example to show how unregulated protein levels can abolish normal cell fate decisions and result in premature ageing or malignancies, depending on the mechanisms involved. RAD51 is one of the most conserved proteins known and essential for cell survival (Henning & Sturzbecher, 2003; Sonoda et al., 1998). While no mutations have ever been detected in human cancers, in many tumors significantly up- or downregulated levels of RAD51 have been observed (Maacke et al., 2000; Henning & Sturzbecher 2003; Klein, 2008). Moreover, high-level expression of RAD51 is an independent prognostic marker of survival in non-small-cell lung cancer patients (Qiao et al., 2005). In addition, haematopoietic progenitor cells, when Rad51 is overexpressed showed elevated levels of chromosomal alterations, similar to those observed in tumors of the hematopoietic system (Francis and Richardson, 2007). Notably, both positive and negative regulation of HRR is required in order to maintain genomic stability with precise repair and suppression of deleterious rearrangements. The only tumorigenesis-related variation found in the *rad51* gene is a G->C change in the 5' untranslated rad51 mRNA region. This variation has been correlated to higher risk for breast cancer in BRCA2 mutation carriers and is possibly involved in mRNA modified translation capability

resulting in abnormal RAD51 protein levels (Antoniou et al., 2007).

Scheme 5. Scematic representation of human Rad51 protein. The areas responsible for

**A B**

BRCA2 binding p53-binding **T301** 

**1 17 96 314 339** 

**179 190**

*Homo sapiens* **Rad51**

P

**Y315 c-Abl** 

P

**Chk1** 

interaction with p53 and BRCA2 are indicated.

evolutionarily conserved. RAD51 replaces RPA in ssDNA, a process regulated by the DNAbinding domain of BRCA2, in cooperation with the BRC repeats and the contribution of other RAD52 epistasis group members as Rad52 and Rad54. BRC repeats of BRCA2 bind to the core of RAD51 by mimicking the structure of an adjacent Rad51 monomer (Pellegrini at al., 2002). RAD51 loading on ssDNA is promoted by the BRCA2[BRC1–8] region while RAD51 assembly on dsDNA is at the same time suppressed. This way the efficiency of RAD51-mediated HRR is further enhanced. RAD51 function can either be stimulated or suppressed by activities of the BRC repeats, depending on the experimental conditions used and the BRC: RAD51 molar ratio used (Galkin et al., 2005; Shivji et al., 2009; Carreira et al., 2009; Rajendra & Venkitaraman, 2010). BRC4 also blocks nucleation of RAD51 onto dsDNA while not disassembling Rad51-dsDNA filaments. (Carreira A, et al., 2009). At lower molar ratios BRC3 or BRC4 actually bind and form stable complexes with RAD51-DNA nucleoprotein filaments. Only at high concentrations of the BRC repeats are filaments disrupted. The specific protein-protein contacts occur in the RAD51 filament by means of the N-terminal domain of RAD51 for BRC3 and the nucleotide-binding core of RAD51 for BRC4 (Galkin et al., 2005; Rajendra & Venkitaraman 2009). These observations show that the BRC repeats bind distinct regions of RAD51 and are nonequivalent in their mode of interaction. These results might explain how disruption of a single RAD51 interaction site in BRCA2 might modulate the ability of RAD51 to promote recombinational repair and lead to an increased risk of breast cancer. Moreover, the dysregulated molar ratio present in a cell may drive hyper-recombination effects leading to abnormal outcome and in part may explain why mutations in BRCA2 predispose individuals to breast cancer, a consequence of the role of BRCA2 in DNA repair.

Scheme 4. Simplistic cartoon of the HRR process depicting Rad51 functions.

evolutionarily conserved. RAD51 replaces RPA in ssDNA, a process regulated by the DNAbinding domain of BRCA2, in cooperation with the BRC repeats and the contribution of other RAD52 epistasis group members as Rad52 and Rad54. BRC repeats of BRCA2 bind to the core of RAD51 by mimicking the structure of an adjacent Rad51 monomer (Pellegrini at al., 2002). RAD51 loading on ssDNA is promoted by the BRCA2[BRC1–8] region while RAD51 assembly on dsDNA is at the same time suppressed. This way the efficiency of RAD51-mediated HRR is further enhanced. RAD51 function can either be stimulated or suppressed by activities of the BRC repeats, depending on the experimental conditions used and the BRC: RAD51 molar ratio used (Galkin et al., 2005; Shivji et al., 2009; Carreira et al., 2009; Rajendra & Venkitaraman, 2010). BRC4 also blocks nucleation of RAD51 onto dsDNA while not disassembling Rad51-dsDNA filaments. (Carreira A, et al., 2009). At lower molar ratios BRC3 or BRC4 actually bind and form stable complexes with RAD51-DNA nucleoprotein filaments. Only at high concentrations of the BRC repeats are filaments disrupted. The specific protein-protein contacts occur in the RAD51 filament by means of the N-terminal domain of RAD51 for BRC3 and the nucleotide-binding core of RAD51 for BRC4 (Galkin et al., 2005; Rajendra & Venkitaraman 2009). These observations show that the BRC repeats bind distinct regions of RAD51 and are nonequivalent in their mode of interaction. These results might explain how disruption of a single RAD51 interaction site in BRCA2 might modulate the ability of RAD51 to promote recombinational repair and lead to an increased risk of breast cancer. Moreover, the dysregulated molar ratio present in a cell may drive hyper-recombination effects leading to abnormal outcome and in part may explain why mutations in BRCA2 predispose individuals to breast cancer, a consequence of

Scheme 4. Simplistic cartoon of the HRR process depicting Rad51 functions.

the role of BRCA2 in DNA repair.

RAD51 filaments are further stabilized by direct interaction of the BRCA2 C terminus to the interface created by two adjacent RAD51 protomers. This way filaments cannot be dissociated by the BRC repeats. Interaction of the BRCA2 C terminus with the RAD51 filament causes a large movement of the flexible RAD51 N-terminal domain that is important in regulating filament dynamics. RAD51 interaction with the BRCA2 C-terminal region may facilitate efficient nucleation of RAD51 multimers on DNA and thereby stimulate recombination-mediated repair. (Esashi et al., 2007). Data from studies the Caenorhabditis elegans BRCA2 homolog CeBRC-2 support a model where an interaction with RAD-51 alone is likely involved in filament nucleation, whereas a second independent interaction is involved in *in situ* stabilization of RAD51 filaments by BRCA2 and provide further insight into why mutations in many different positions within BRCA2 lead to loss of genomic stability (Petalcorin et al., 2007).

RAD51, the central homology strand search and strand exchange effector in HRR can serve as a nice example to show how unregulated protein levels can abolish normal cell fate decisions and result in premature ageing or malignancies, depending on the mechanisms involved. RAD51 is one of the most conserved proteins known and essential for cell survival (Henning & Sturzbecher, 2003; Sonoda et al., 1998). While no mutations have ever been detected in human cancers, in many tumors significantly up- or downregulated levels of RAD51 have been observed (Maacke et al., 2000; Henning & Sturzbecher 2003; Klein, 2008). Moreover, high-level expression of RAD51 is an independent prognostic marker of survival in non-small-cell lung cancer patients (Qiao et al., 2005). In addition, haematopoietic progenitor cells, when Rad51 is overexpressed showed elevated levels of chromosomal alterations, similar to those observed in tumors of the hematopoietic system (Francis and Richardson, 2007). Notably, both positive and negative regulation of HRR is required in order to maintain genomic stability with precise repair and suppression of deleterious rearrangements. The only tumorigenesis-related variation found in the *rad51* gene is a G->C change in the 5' untranslated rad51 mRNA region. This variation has been correlated to higher risk for breast cancer in BRCA2 mutation carriers and is possibly involved in mRNA modified translation capability resulting in abnormal RAD51 protein levels (Antoniou et al., 2007).

Scheme 5. Scematic representation of human Rad51 protein. The areas responsible for interaction with p53 and BRCA2 are indicated.

Structure-Function Relationship of DNA Repair

unpublished data).

Proteins: Lessons from BRCA1 and RAD51 Studies 121

Fig. 6. MCF7 cells expressing exogenous Rad51wt-EYFP and Rad51m6-EYFP as indicated. Endogenous BRCA1 expression, as detected by immunofluoresence, shows a significant reduction in all cells expressing the Rad51m6-EYFP in comparison to both

the Rad51wt-EYFP expressing as well as to non-transfected cells (Boutou et al.,

serve as a potential anti-cancer agent, selectively targeting malignant cells.

cells, but do not affect the Caspase 3 defective MCF-7 cells.

Moreover cells expressing RAD51m6 showed a modification in their cell cycle progression (data not shown) accompanied by modifications in expression of BRCA1 (fig 6), p53 and p21waf1 (data not shown). Notably, RAD51m6 electively kill certain cancer cell lines as HeLa

Double strand breaks (DSBs) of DNA is the most deleterious damage of the genome since if not repaired accurately can result in ICL, translocations, chromatin rearrangements, LOH and mutation accumulation. HRR restores DNA damage in mitotic cells by gene conversion, where the broken sequence is converted to the sequence of the repair template (original sequence), which remains unaltered. In case of HRR misregulation other templates can be used including homologous chromosomes and repetitive elements on heterologous chromosomes. Such data indicate that single amino acid residue alterations of Rad51 are capable to modify the behaviour of the entire protein, presumably through structural modifications. These results, combined with the fact that RAD51 protein in nature is not mutated, suggest that its proper function is strongly dependent on its high degree of structural conservation throughout evolution. Moreover, the combination of the active peptide of such mutants under the promoter of human RAD51 which in the absence of the N-terminal region of Rad51 enhances expression up to 10 fold in cancer cell lines, could

Since Rad51 overexpression can compensate for loss of function of other key molecules of DDR, including BRCA1 and BRCA2, experimental evidences from various research groups support two models: 1. Rad51 abnormal levels lead to genomic instability early in cancer development, thereby placing Rad51 modified expression as a leading cause of transformation and 2. Rad51 overexpression can protect cancer cells from DNA damage as more effective repair occurs further stabilizing the neoplastic clone and render it more aggressive and metastatic (Schild & Wiese, 2010). As cancer is an extremely complex set of diseases and can develop by different aetiologies while achieving similar phenotype of independent and uncontrolled growth the two models presented by Schild and Wiese can each adequately explain the neoplastic procedure of different cancer types.

The exact causes of Rad51 overexpression are still poorly explored but there is a number of data indicating both transcriptional regulation and protein stability and turnover modification. p53, the tumor suppressor that is implicated in DNA repair control, is involved in transcriptional regulation of *rad51* (Arias-Lopez et al., 2006). p53 is mutated in about half of human cancers resulting in loss of suppression of rad51 transcriptional regulation. Notably, as p53 directly interacts with Rad51 (Stürzbecher et al., 1996), in cases of p53 mutations inhibition of Rad51 activity could be abolished. Moreover, in cases of either TP53 deletion or some TP53 point mutations Rad51 expression up-regulation is detected. The Transcriptional activator protein 2 (AP2), in combination with p53 suppresses rad51 transcription (Hannay et al., 2007). Abl kinase phosphorylates Rad51 in Tyr315 and in cases of the presence of the oncogenic constituvely active BCR/Abl fusion tyrosine kinase (i.e. in Ph+ leukaemias) Rad 51 expression is increased (Slupianek et al., 2001).

Aiming in further clarifying aspects of structure-function relationship of RAD51, we produced several RAD51 mutants by altering amino acid residue candidates to be involved in RAD51-BRCA2 or RAD51-p53 interaction (fig 5). Exogenous expression the RAD51m6 mutant, fused to EYFP, altered their subcellular localization compared to the wt protein.

Fig. 5. Solved structure of Rad51-BRC4 complex (adapted from PDB: 1NOW, (Pellegrini et al., 2002)) where candidate residues presumed to alter Rad51-BRCA2/p53 complex interaction are indicated. *In vitro* Site-Directed mutagenesis was employed in order to alter each indicated residue to Ala. Mutant form positions are indicated.

Since Rad51 overexpression can compensate for loss of function of other key molecules of DDR, including BRCA1 and BRCA2, experimental evidences from various research groups support two models: 1. Rad51 abnormal levels lead to genomic instability early in cancer development, thereby placing Rad51 modified expression as a leading cause of transformation and 2. Rad51 overexpression can protect cancer cells from DNA damage as more effective repair occurs further stabilizing the neoplastic clone and render it more aggressive and metastatic (Schild & Wiese, 2010). As cancer is an extremely complex set of diseases and can develop by different aetiologies while achieving similar phenotype of independent and uncontrolled growth the two models presented by Schild and Wiese can each adequately

The exact causes of Rad51 overexpression are still poorly explored but there is a number of data indicating both transcriptional regulation and protein stability and turnover modification. p53, the tumor suppressor that is implicated in DNA repair control, is involved in transcriptional regulation of *rad51* (Arias-Lopez et al., 2006). p53 is mutated in about half of human cancers resulting in loss of suppression of rad51 transcriptional regulation. Notably, as p53 directly interacts with Rad51 (Stürzbecher et al., 1996), in cases of p53 mutations inhibition of Rad51 activity could be abolished. Moreover, in cases of either TP53 deletion or some TP53 point mutations Rad51 expression up-regulation is detected. The Transcriptional activator protein 2 (AP2), in combination with p53 suppresses rad51 transcription (Hannay et al., 2007). Abl kinase phosphorylates Rad51 in Tyr315 and in cases of the presence of the oncogenic constituvely active BCR/Abl fusion tyrosine kinase

Aiming in further clarifying aspects of structure-function relationship of RAD51, we produced several RAD51 mutants by altering amino acid residue candidates to be involved in RAD51-BRCA2 or RAD51-p53 interaction (fig 5). Exogenous expression the RAD51m6 mutant, fused to EYFP, altered their subcellular localization compared to the wt protein.

**m2** 

**m3** 

**m6 m8** 

**m9** 

**m5** 

Fig. 5. Solved structure of Rad51-BRC4 complex (adapted from PDB: 1NOW, (Pellegrini et al., 2002)) where candidate residues presumed to alter Rad51-BRCA2/p53 complex

interaction are indicated. *In vitro* Site-Directed mutagenesis was employed in order to alter

each indicated residue to Ala. Mutant form positions are indicated.

(i.e. in Ph+ leukaemias) Rad 51 expression is increased (Slupianek et al., 2001).

explain the neoplastic procedure of different cancer types.

Fig. 6. MCF7 cells expressing exogenous Rad51wt-EYFP and Rad51m6-EYFP as indicated. Endogenous BRCA1 expression, as detected by immunofluoresence, shows a significant reduction in all cells expressing the Rad51m6-EYFP in comparison to both the Rad51wt-EYFP expressing as well as to non-transfected cells (Boutou et al., unpublished data).

Moreover cells expressing RAD51m6 showed a modification in their cell cycle progression (data not shown) accompanied by modifications in expression of BRCA1 (fig 6), p53 and p21waf1 (data not shown). Notably, RAD51m6 electively kill certain cancer cell lines as HeLa cells, but do not affect the Caspase 3 defective MCF-7 cells.

Double strand breaks (DSBs) of DNA is the most deleterious damage of the genome since if not repaired accurately can result in ICL, translocations, chromatin rearrangements, LOH and mutation accumulation. HRR restores DNA damage in mitotic cells by gene conversion, where the broken sequence is converted to the sequence of the repair template (original sequence), which remains unaltered. In case of HRR misregulation other templates can be used including homologous chromosomes and repetitive elements on heterologous chromosomes. Such data indicate that single amino acid residue alterations of Rad51 are capable to modify the behaviour of the entire protein, presumably through structural modifications. These results, combined with the fact that RAD51 protein in nature is not mutated, suggest that its proper function is strongly dependent on its high degree of structural conservation throughout evolution. Moreover, the combination of the active peptide of such mutants under the promoter of human RAD51 which in the absence of the N-terminal region of Rad51 enhances expression up to 10 fold in cancer cell lines, could serve as a potential anti-cancer agent, selectively targeting malignant cells.

Structure-Function Relationship of DNA Repair

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### **4. Conclusions**

In conclusion maintenance of genome integrity depends on structure-function relationship of the protein molecules involved. Proper response to DNA damage mainly relies on functional components of DDR driving their appropriate complex formation with partner proteins. These processes are regulated by a number of post-translational modifications, distinct protein isoforms and protein availability (stability / degradation). In case these interactions are deregulated due to genetic / epigenetic causes a balanced cell cycle progression and cell fate determination are abolished in favour of cancer/ageing. Structural / biophysical data accompanied by functional experiments of key DNA repair molecules are significant for: (a) elucidating which residues or structural elements are really necessary for proper function at the molecular level, (b) asses/classify variants identified in individuals, (c) enriching diagnostic markers in cancer and (d) designing effective small molecules to target protein molecules essential for cell survival and genome integrity.

Co-operation of various disciplines is a fundamental prerequisite for fulfilling such a vision, and numerous attempts worldwide work on this subject with promising results.

#### **5. References**


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Co-operation of various disciplines is a fundamental prerequisite for fulfilling such a vision,

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target protein molecules essential for cell survival and genome integrity.

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and numerous attempts worldwide work on this subject with promising results.

**4. Conclusions** 

**5. References** 

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*Repair and Mutagenesis, part 3.* ASM Press. 2nd edition


**8** 

*China* 

Yulin Zhang and Dexi Chen

*Department of infectious diseases, Beijing You'an Hospital* 

**The Involvement of E2F1 in the Regulation of** 

**XRCC1-Dependent Base Excision DNA Repair** 

Reactive oxygen species, ionizing radiation and alkylating agents can attack on DNA resulting in single or double strand breaks, generation of abasic sites, base and sugar lesions [1]. Double-strand breaks (ds breaks) are repaired by two different types of mechanism. One type takes advantage of proteins that promote homologous recombination (HR) to obtain instructions from the sister or homologous chromosome for proper repair of breaks. The other type permits joining of ends even if there is no sequence similarity between them. The latter process is called non-homologous end joining (NHEJ).The process by which complex single-strand breaks (those that cannot be directly religated) are repaired (SSBR) in some ways resembles NHEJ. Here we shall mainly discuss the mechanism of base excision repair

The major pathway to remove damaged DNA bases is Base Excision Repair (BER, *Fig. 1*). BER can be divided into five steps: (i) excision of damaged base by the specific DNA glycosylase and formation of apurinic/apyrimidinic (AP) site; (ii) cleavage of phosphodiester bond at AP site by AP-endonuclease or AP-lyase; (iii) removal of chemical

The first step of the BER pathway is recognition of damaged base by the specific DNA glycosylase, which cleaves N-glycosidic bond leaving behind a free base and an AP site. In humans about 10 DNA glycosylases of different, but partially overlapping substrate specificities are known [3]. Some of them are bifunctional enzymes, which have endowed AP-lyase activity and cleave phosphodiester bonds at 3' side of AP site either by ß- or ß/δ elimination. *E. coli* endonuclease III (Nth), its human homolog, hNTH1 and human 8-oxoG DNA glycosylase (OGG1) catalyse reaction of ß- elimination, which creates alpha/ßunsaturated aldehyde (3'dRP) at the 3' end of cleaved DNA strand. Bacterial formamidopyrimidine DNA glycosylase (Fpg), endonuclease VIII (Nei) and two human homologs of the latter, NEIL1 and NEIL2 catalyse ß/ δ-elimination and remove deoxyribose residue leaving phosphate at the 3' end of cleaved DNA strand. Monofunctional DNA glycosylases need the assistance of AP-endonucleases, which hydrolyse phosphodiester bond at the 5' end of the AP site. This yields DNA single strand break (SSB) with the 5'end

groups interfering with gap filling and ligation; (iv) gap filling; (v) ligation [2].

**1. Introduction** 

(BER) of SSBR.

**2. Base excision DNA repair** 

*Beijing liver disease research institute, Capital Medical University, Beijing* 

