**Structure-Function Relationship of DNA Repair Proteins: Lessons from BRCA1 and RAD51 Studies**

Effrossyni Boutou1,2, Vassiliki Pappa3, Horst-Werner Stuerzbecher4 and Constantinos E. Vorgias1 *1Department of Biochemistry & Molecular Biology Faculty of Biology, School of Sciences Athens University, Athens 2Prenatal Diagnosis Lab, Laiko Hospital, Athens 32nd Propaedeutic Pathology Clinic, Medical School, Athens University, Athens 4Molecular Cancer Biology Group, Institute of Pathology, UK-SH, Luebeck 1,2,3Greece 4Germany* 

### **1. Introduction**

102 DNA Repair

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Accurate transfer of genetic information is vital for all living organisms in order to guarantee species survival. DNA damage occurs spontaneously during a cell's life due to either endogenous causes such as Reactive Oxygen Species (ROS) produced during metabolism or due to exogenous insults such as Ionizing Radiation (IR) or genotoxic agents in food / water and environment, to which an organism is exposed. Endogenous damage, due to intrinsic instability of chemical bonds in DNA structure, occurs spontaneously under normal physiologic conditions and is calculated to be approximately 104 events per cell, per day (Lindahl, 1993). Moreover, during DNA replication base adducts can cause collapse of replication forks and DNA double strand breaks (DSBs) are introduced in order to reinitiate genome duplication process.

As the genome carries all necessary information for life and evidently preservation of genome integrity is critical for cell survival, a number of mechanisms have evolved over time to ensure the most effective performance of the genome repair procedure. DNA repair mechanisms are capable of repairing practically all different types of chromosomal lesions (single and double strand breaks, base modifications, etc.) ensuring that genetic information is accurately transferred to the next generation. The cell's response to DNA damage (DNA Damage Response, DDR) encompasses a complex network of proteins, consisting of DNA damage recognition, signal transduction, transcriptional regulation, cell cycle control, DNA repair and verification of the repair efficiency, depending on the type of lesion, the replication status of the genome as well as the cell cycle stage. (scheme 1). Many excellent recent reviews as well as other chapters in the current volume extensively cover this topic (Rogakou, 1999; Lisby & Rothstein 2005; Murphy & Moynahan, 2010).

Defects in repair efficiency are the consequence of dysfunction of either upstream damage signalling or the central repair process. The current chapter covers topics referring to

Structure-Function Relationship of DNA Repair

**2. Consequences of genomic instability** 

**2.1 Genomic instability and cancer** 

processes.

Proteins: Lessons from BRCA1 and RAD51 Studies 105

supported by its correlation with increased breast & ovarian familial cancer susceptibility in individuals carrying BRCA1 mutations. On the other hand, RAD51 is a relatively small and rigid protein playing a basic role (homology search strand and strand exchange) in the high fidelity DNA repair mechanism of Homologous Recombination (HR). RAD51 appears absolutely vital for cell survival, as its depletion results in embryonic lethality, it is highly conserved throughout evolution and up to now there is not a single mutation in the amino acid sequence detected in any type of cancer, although there is a strong correlation between

Loss or insufficiency of DDR and genome repair can lead to an increased susceptibility to cancer due to the consequential genomic instability. Ineffective repair may result in subsequent mutations of genes required for cellular replication and division. The genome repair pathways also communicate with processes involved in induction of senescence and apoptosis when the damage cannot be repaired. Carefully balanced signalling cascades and regulatory systems are implicated in the maintenance of healthy cell survival in order to unfavour tumorigenesis and maintain stem and progenitor cells for renewal (anti-ageing) (Seviour & Lin 2010). Therefore, an effectively repaired genome is crucial not only for cancer prevention but also for lifespan extension. This notion is even more enhanced by the emerging benefit of the response of HR defective tumors to double strand break (DSB) producing therapies a promising and continuously evolving field. A clearer understanding of the biochemical, structural and genetic processes in conjuction with clinical data will lead to the development of more effective treatment strategies for both cancer and ageing

It is generally accepted that tumors are derived from a single genetically unstable cell, and that the unstable cell population as a whole continues to acquire further chromosomal abnormalities over time, although the precise mechanisms of acquisition of these abnormalities still remain unclear. Hereditary cancers are often characterized by the presence of a specific type of genomic instability, termed chromosomal instability. In these cancers, chromosomal instability can often be attributed to mutations in DNA repair genes, suggesting that the driving force behind tumor development is an increase in spontaneous genetic mutations resulting from lack of appropriate management of DNA damage. A second form of genomic instability, termed microsatellite instability, is also associated with defects in DNA repair, namely the mismatch repair system. However, in non-hereditary sporadic tumors, the picture is less clear. It should be emphasized that cancer is an extremely complex set of diseases, and that cancer cells develop many different mechanisms to achieve a similar phenotype of independent and uncontrolled growth (Hanahan &

Many of the DDR components including BRCA1 are known to be lost or mutated in human tumors. While loss of BRCA1 has been shown to lead to the development of mammary tumors in mouse models, the genetic diversity within those tumors suggests that the loss of BRCA1 may not directly be responsible for tumorigenesis. It is more likely, therefore, that the role of BRCA1 in the initiation of cancer is a result of its effects on DNA repair and the maintenance of genomic integrity. BRCA1 -/- tumors are shown to display numerous

Weinberg, 2000; Luo et al., 2009 as reviewed by Schild & Wiese 2010).

its expression levels and both cancer development and cancer progression.

Scheme 1. Simplified diagram of DNA damage response network

factors/events influencing the structure - function relation of key molecules involved in each of the two processes, namely the BRCA1 and RAD51/Rad51 proteins, respectively. The breast cancer susceptibility gene 1 (brca1), isolated by reverse genetics in 1994, encodes for a large multifunctional protein (BRCA1) whose function is regulated by multiple posttranslational modification events, driving the multi tasks performed, by which BRCA1 conducts almost all steps of DDR. The important anti-tumorigenic role of BRCA1 is strongly

**Exogenous causes** • Ionizing radiation

• Environmental mutagens, etc.

**Base modifications Repaired by: BER, etc** 

> **MisMatch Repair**

> > **cancer**

**aging** 

**Ineffective Repair / LOH / Gene rearrangement or translocation** 

**apoptosis** 

**DNA** 

**Double Strand Breaks (most deleterious)** 

**Mainly Repaired by:** 

**Homologous Recombination (cell cycle phase dependent (S/G2), error free)** 

**Endogenous causes** • Metabolism (ROS),

**Single Strand Breaks** 

**Non-Homologous End Joining (cell cycle phase independent, error prone)** 

• Stalled replication forks, etc.

Scheme 1. Simplified diagram of DNA damage response network

**Effective Repair** 

**Cell cycle progression, cell proliferation** 

factors/events influencing the structure - function relation of key molecules involved in each of the two processes, namely the BRCA1 and RAD51/Rad51 proteins, respectively. The breast cancer susceptibility gene 1 (brca1), isolated by reverse genetics in 1994, encodes for a large multifunctional protein (BRCA1) whose function is regulated by multiple posttranslational modification events, driving the multi tasks performed, by which BRCA1 conducts almost all steps of DDR. The important anti-tumorigenic role of BRCA1 is strongly supported by its correlation with increased breast & ovarian familial cancer susceptibility in individuals carrying BRCA1 mutations. On the other hand, RAD51 is a relatively small and rigid protein playing a basic role (homology search strand and strand exchange) in the high fidelity DNA repair mechanism of Homologous Recombination (HR). RAD51 appears absolutely vital for cell survival, as its depletion results in embryonic lethality, it is highly conserved throughout evolution and up to now there is not a single mutation in the amino acid sequence detected in any type of cancer, although there is a strong correlation between its expression levels and both cancer development and cancer progression.
