![]() ![]() These DNA gaps have been shown to strongly inhibit NHEJ ( 10). DSBs with 3′ overhangs retain base-pairing potential after resection begins, but resection introduces extended gaps adjacent to the terminal nucleotides ( Fig. RPA also stimulates Exo1 and directs DNA2 cleavage to the 5′ flap after unwinding by BLM ( 6, 9).Īt DSBs with 5′ overhangs, resection eliminates the ability to align the ends for precise NHEJ ( Fig. The single-stranded DNA (ssDNA) binding protein RPA (replication protein A) coats the 3′ tails generated during resection, preventing the formation of secondary structures ( 8). There is evidence of cross talk between these pathways, as MRN and BLM both stimulate Exo1 in vitro ( 6, 7). MRX is required for the initiation of resection near the DSB, whereas Exo1 and Dna2 carry out long-range resection. As first revealed in genetic studies performed with the budding yeast Saccharomyces cerevisiae, three distinct nucleases function in the DSB end resection process: the 5′-to-3′ exonuclease I (Exo1), the flap endonuclease Dna2 in conjunction with the RecQ family helicase Sgs1 (BLM in humans), and Mre11, part of the Mre11-Rad50-Xrs2 (MRX) complex (MRE11-RAD50-NBS1, or MRN, in humans) ( 4, 5). It has become clear that whether or not the DNA ends have undergone extensive 5′-to-3′ nucleolytic resection exerts a major impact on this choice, such that ends that bear long 3′ DNA tails become destined for HR repair ( 2, 3). How DSB repair pathway choice is determined at the molecular level has been the subject of intense study for quite some time. This process is normally accurate but requires that cells be in the S or G 2 phase of the cell cycle, when DNA replication generates the sister chromatid to direct the repair process. In HR, the intact sister chromatid is most often engaged as the information donor. Moreover, when ends from two different chromosomes are joined, a chromosomal translocation ensues. ![]() In the latter case, joining is associated with DNA sequence loss. While NHEJ accurately repairs “clean” DSBs whose ends are compatible and harbor undamaged terminal nucleotides, it is also capable of joining mismatched termini or termini that harbor damaged, otherwise-unligatable terminal nucleotides. ![]() NHEJ is active throughout the cell cycle. NHEJ entails the tethering of the broken DNA ends and their ligation ( 1). Two mechanistically distinct pathways have evolved to eliminate DSBs from the genome: nonhomologous DNA end joining (NHEJ) and homologous recombination (HR), both of which are conserved in all kingdoms of life. Failure to accurately repair DSBs can lead to gross chromosome rearrangements or mutations at the break site, which can cause cell death, cell transformation, and tumorigenesis. ![]() Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G 1 to homologous recombination in S/G 2.ĭNA double-strand breaks (DSBs) are exceedingly dangerous chromosomal lesions. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. Specifically, nonhomologous end joining is the predominant mechanism used in the G 1 phase of the cell cycle, while homologous recombination becomes fully activated in S phase. When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. ![]()
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