Overview of Base-Excision and Homologous recombination repair − Rakyat Biologi

In all human cells, DNA is subjected to frequent damage secondary to environmental insults, toxic metabolites and DNA replication errors. Single-stranded breaks (SSBs) are one of the most frequent mechanisms of damage and can occur at a rate as high as 10 000 per day (1). A SSB is defined as a loss on continuity in the deoxyribose sugar backbone in one strand of the DNA double helix and can be potentially accompanied by the loss of the nucleotide base at the site of the break (2). The most common causes of SSB are: 1) reactive oxygen species generated by cellular metabolism (2); 2) failure of DNA base-excision repair (BER) to completely repair damaged or absent nucleotide bases (3); and, 3) failure of DNA topoisomerase I to resolve cleavage complexes generated during gene transcription and DNA replication, leaving a break in the deoxyribose sugar backbone that was initially generated by the creation of the complex (4).

Detection of SSBs, and subsequent activation of BER, is thought to require the activity of PARP enzymes. Of the 17 known family members, PARP-1 has been the most extensively evaluated and appears to play the most important role. Figure 1 provides an overview of the role that PARP is presumed to play in BER. Found in the nucleus, PARP-1 recognizes SSBs in DNA via two zinc-fingers which are structurally homologous to the DNA binding sites in DNA ligase III and 3’ DNA phosphoesterase, two other DNA repair enzymes (5). Upon binding to a SSB, PARP-1 undergoes a conformation shift, activating its catalytic capabilities, leading to the synthesis of a poly (ADP-ribose) (PAR) polymer to itself, histones and other nuclear proteins using nicotinamide adenine dinucleotide as the substrate. The PAR polymer then serves as a signal to recruit other key enzymes in the BER repair process, such as DNA ligase III, DNA polymerase beta (Pol-beta) and X-ray cross-complementing gene 1 (XRCC1) (6). In particular, recruitment of XRCC1 appears to be critical as it acts as the primary scaffold protein upon which the BER complex is assembled (7). In addition, the attachment of PAR to histones H1 and H2B relaxes the chromatin structure, facilitating the repair process (8). PARP-2 has been shown to interact with PARP-1 and independently contribute to the BER complex recruitment process (9). Once the PAR polymer has been synthesized and repair completed, PARP-1 dissociates from DNA and the PAR chains are degraded by poly(ADP-ribose) glycohydrolase (PARG), resetting PARP-1 to its inactive conformation and restoring the chromatin structure (10).

Failure to repair SSBs results in DNA double-stranded breaks (DSBs) as replication forks encountering SSBs either stall or collapse (11). The two major DSB repair pathways within the cell are HR and non-homologous end-joining (NHEJ). HR represents a largely error-free mechanism to repair DSBs as homologous duplex DNA is used as a template for repair DNA synthesis; in contrast, NHEJ promotes the direct ligation of DSB ends, potentially resulting in insertions, deletions, base-substitutions and translocations if different components of the genome are brought together (12). Because NHEJ is significantly error-prone, DSBs represent a significant risk to overall genomic integrity and a significant threat to cellular viability (13).

A simplified schematic of DSB repair is presented in Figure 2. NHEJ is active throughout the cell cycle and is facilitated by 53BP1. 53BP1 interferes with end-resection activity of MRE11/RAD50/NBS1 (MRN) complex, directing the cell towards NHEJ repair of a DSB (12). In contrast, HR activity needs to be restricted to the S/G2 phase of the cell cycle as an intact sister chromatid is required to serve as a template for repair. In human cells, restriction of HR to S/G2 occurs through three primary mechanisms. First, BRCA1 expression is tightly regulated and begins to increase in late G1 and S phase with peak levels detected during G2 (14). Second, protein levels of

CtIP, an important activator of HR, remain low due to proteasome-mediated degradation until S/G2; at this point in time, this degradation becomes inhibited, allowing for CtIP accumulation within the nucleus (15). Third, cyclin-dependent kinases (CDK) 1 and 2, which are most active during S/G2, phosphorylates CtIP and BRCA1, resulting in their activation (15-17). Activation of BRCA1 prevents 53BP1 from interfering with MRN, permitting end-resection and creating the single-stranded sequences (ssDNA) at the DSB necessary to initiate HR repair (18). Replication Protein A (RPA) is then added to the ssDNA to prevent the formation of secondary structures (19). Following end-resection, BRCA2 facilitates RAD51 loading which leads to strand-invasion and DNA repair in an error-free process (12, 20-23).

Cell cycle arrest is another crucial aspect of the HR repair process. Without arrest, cell cycle progression will lead to mitosis and loss of the sister chromatids necessary for HR repair. The Ataxia Telangiectasia-Mutated (ATM) protein, in conjunction with MRN complex, is recruited and activated in response to DSBs (24). Activated ATM interacts and phosphorylates multiple proteins involved in initiating repair and checkpoint arrest, including Checkpoint kinase 2 (Chk2), NBS1, BRCA1 and MDC1 (25). Activation of these proteins leads to cell cycle arrest through actions on p53, Cdc25, BRCA1, FOXM1 and E2F1 (26). Loss of the ATM-Chk2 pathway has been shown to lead to genomic instability within cells as loss of cell cycle arrest limits HR repair of DNA damage, leading to accumulation of mutations and genomic instability as NHEJ assumes responsibility for repair.