**4.5 PARP family**

62 DNA Repair

co-localize at the centrosome. This interaction may be important for the DNA mitotic

About 10% of women diagnosed with breast cancer have inherited mutations in *BRCA1* or *BRCA2* (Irminger-Finger and Jefford, 2006). Both *BRCA1* and *BRCA2*, products of the familial breast cancer susceptibility gene, are involved in several cellular functions, such as DNA repair, transcriptional regulation, cell-cycle checkpoints, and centrosome maintenance. BRCA1 forms a heterodimer complex with BRCA1-associated RING domain (BARD1), which functions as an E3 ubiquitin ligase. Both BRCA1 and BARD1 contain a RING domain, which mediates DNA–protein and protein–protein interactions, a nuclear export signal sequence at their N-terminus, and tandem BRCT (BRCA1 carboxy-terminal) domains. The BRCA1–BARD1 complex monoubiquitylates γ-tubulin at Lysine 48 and Lysine 344, and overexpression of mutated γ-tubulin at the K48 ubiquitination site results in centrosome amplification and aberration of microtubule nucleation (Starita et al., 2005; Simons et al., 2006). Overexpression of mutated γ-tubulin (K344R) results in an aberration of microtubule nucleation only, suggesting that BRCA1 controls centrosome function by monoubiquitination of γ-tubulin. The BRCA1–BARD1 complex also ubiquitylates the nucleolar phosphoprotein nucleophosmin (NPM also known as B23), which functions in nucleolar organization, cell-cycle regulation, and centrosome duplication. The BRCA1–BARD1 complex polyubiquitinates NPM, leading to its degradation. Aurora A, which localizes at the centrosome and is an important factor for mitotic progression, phosphorylates BRCA1, which contributes to regulation of centrosome duplication. Furthermore, the BRCA1–BARD1 complex regulates microtubule organization

A BRCA2 mutation is involved in approximately 50% of hereditary breast cancers (Yoshida and Miki, 2004). BRCA2 has no sequential or structural similarity with either BRCA1 or BARD1 and localizes at the centrosome. Interaction of BRCA2 with plectin, a cytoskeletal cross-linker protein, is necessary for centrosome anchoring to the nucleus (Niwa et al., 2009). BRCA2 also forms a complex at the centrosome with NPM and ROCK2, an effector of Rho small GTPase. A definite BRCA2 deletion can abrogate the association of BRCA2 with NPM, and cells expressing this deletion mutant show centrosome amplification (Wang et al., 2011), suggesting that the BRCA2–NPM complex maintains centrosome duplication and controls

NBS, which is caused by an *NBS1* gene mutation, is characterized by growth retardation, a birdlike face, immunodeficiency, predisposition to malignancy, and microcephaly (Matsuura et al., 1998). NBS patient cells have a defect in the cell-cycle checkpoint and hyper-radiosensitivity. NBS1 is a multifunctional protein that participates in homologous recombination repair, DNA replication, the cell-cycle checkpoint, and apoptosis (Tauchi et al., 2002). NBS1 forms a complex with MRE11 and RAD50 (MRN complex), and this complex is required for recruitment of ATM to DNA damage sites and for efficient phosphorylation of ATM substrates (Iijima et al., 2008). NBS1 contains a forkhead-associated (FHA) domain and a BRCT domain at the N-terminus, the binding motif for MRE11, ATM, and RNF20, which is a E3 ubiquitin ligase for H2B, at the C-terminus (Nakamura et al., 2011). The NBS1 FHA domain is required for ATR interaction (Shimada et al., 2009). NBS1

damage-dependent checkpoint, although the details remain unknown.

**4.3 BRCA1 and BRCA2** 

through a Ran-dependent import pathway.

cell division.

**4.4 NBS1** 

PARP1 catalyzes the formation of long branched polyADP-ribosylation covalently attached to target proteins using NAD+ as a substrate. Many proteins are poly(ADP-ribosyl)ated by PARP1, and this modification may be involved in transcriptional regulation and DNA repair (Miwa and Masutani, 2007). PARP1–/– mouse cell lines show centrosome amplification (Kanai et al., 2003). Other PARP family proteins, such as PARP3 and tankylase (also known as PARP5a), localize at the centrosome (Smith and de Lange, 1999; Augustin et al., 2003). These reports suggest that PARP family proteins are involved in the control of centrosome duplication.

### **4.6 RAD51 paralogs**

RAD51 and five paralogs, RAD51B (RAD51L1), RAD51C (RAD51L2), RAD51D (RAD51L3), XRCC2, and XRCC3, play important roles in homologous recombination (HR) repair (Date et al., 2006; Renglin Lindh et al., 2007; Cappelli et al., 2011). These proteins have a consensus domain including Walker A and B ATPase domains and are necessary for chromosome stability and the control of chromosome segregation. In mammalian cells, XRCC2 forms a complex with RAD51B and RAD51C, and XRCC3 forms a complex with RAD51C. The XRCC2 complex is involved in the RAD51 loading step to ssDNA in HR repair. The XRCC3 complex is involved in Holliday junction resolution. Loss of RAD51, RAD51B, RAD51C, RAD51D, XRCC2, or XRCC3 leads to centrosome amplification and chromosome instability. RAD51C, XRCC2, or XRCC3-deficient cell lines show centrosome amplification in the M phase, but only XRCC2-deficient cell lines show centrosome amplification at interphase (Renglin Lindh et al., 2007), suggesting that RAD51C and XRCC3, but not XRCC2, may be involved in the same centrosome duplication pathway.

### **4.7 Nonhomologous end-joining repair proteins**

Nonhomologous end-joining (NHEJ) repair proteins such as DNA-PKcs also localize at centrosomes (Zhang et al., 2007). Our previous reports showed that DNA-PKcs-deficient cell lines (SCID) have a slightly increased centrosome number compared to wild-type cell lines. Moreover, another NHEJ factor, Ku70, found in a Ku70-deficient cell line also has a slight increase in centrosome number compared to complementary cell lines (Shimada et al., 2010), indicating that NHEJ factors may be involved in centrosome functions different from HR factors.
