Shared Genetic Pathways Contribute to the Tolerance of Endogenous and Low-Dose Exogenous DNA Damage in Yeast

DNA damage that escapes repair and blocks replicative DNA polymerases is tolerated by bypass mechanisms that fall into two general categories: error-free template switching and error-prone translesion synthesis. Prior studies of DNA damage responses in Saccharomyces cerevisiae have demonstrated that repair mechanisms are critical for survival when a single, high dose of DNA damage is delivered, while bypass/tolerance mechanisms are more important for survival when the damage level is low and continuous (acute and chronic damage, respectively). In the current study, epistatic interactions between DNA-damage tolerance genes were examined and compared when haploid yeast cells were exposed to either chronic ultraviolet light or chronic methyl methanesulfonate. Results demonstrate that genes assigned to error-free and error-prone bypass pathways similarly promote survival in the presence of each type of chronic damage. In addition to using defined sources of chronic damage, rates of spontaneous mutations generated by the Pol ζ translesion synthesis DNA polymerase (complex insertions in a frameshift-reversion assay) were used to infer epistatic interactions between the same genes. Similar epistatic interactions were observed in analyses of spontaneous mutation rates, suggesting that chronic DNA-damage responses accurately reflect those used to tolerate spontaneous lesions. These results have important implications when considering what constitutes a safe and acceptable level of exogenous DNA damage.     

跨损伤合成的DNA聚合酶——一类新的DNA聚合酶

细胞虽然拥有多种修复途径,但有些DNA损伤仍不可避免地会逃避修复而在基因组上保留下来,细胞跨损伤DNA合成的分子机制一直是DNA修复中主要的未解决问题之一.最近通过对一类结构相关性UmuC/DinB蛋白质超家族成员的研究发现它们具有DNA聚合酶功能.这类新发现的DNA聚合酶不同于经典的复制性DNA聚合酶,它们能以易误/突变(error-prone/mutagenic)或无误(error-free)方式进行跨损伤(translesion)DNA合成,并且从细菌到人在进化上功能保守.     

Effect of DNA sequence and nucleotide cofactors on hRad51 binding to ssDNA: role of hRad52 in recruitment

hRad51 binding to ssDNA is significantly lowered in the presence of a nucleotide cofactor ATP/ADP/ATPgammaS. In these conditions, presence of trace amounts of hRad52 protein restores hRad51 binding to DNA. In the absence of any nucleotide cofactor where intrinsic binding of hRad51 to ssDNA is higher, hRad52 brings about no improved binding. hRad51 binding to ssDNA is strongly influenced by the DNA sequence. The protein binding to repeat sequences is poor compared to that of mixed DNA sequence. Interestingly, presence of hRad52 restores the ability of hRad51 binding to such DNA targets as well. Moreover, all the cooperative effects of hRad52 on hRad51 binding are highly specific to the latter's binding to ssDNA and not to dsDNA. These results help us to model important mechanistic steps of hRad51 presynapsis on ssDNA templates.     

Rad51 replication fork recruitment is required for DNA damage tolerance

Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non‐repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non‐DSB DNA lesions, presumably single‐stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle‐regulated replicative and repair functions.     

From strand exchange to branch migration; bypassing of non-homologous sequences by human Rad51 and Rad54

Rad51 and Rad54 play crucial roles during homologous recombination. The biochemical activities of human Rad51 (hRad51) and human Rad54 (hRad54) and their interactions with each other are well documented. However, it is not known how these two proteins work together to bypass heterologous sequences; i.e. mismatched base pairs, during homologous recombination. In this study, we used a fluorescence resonance energy transfer assay to monitor homologous recombination processes in real time so that the interactions between hRad54 and hRad51 during DNA strand exchange and branch migration, which are two core steps of homologous recombination, could be characterized. Our results indicate that hRad54 can facilitate hRad51-promoted strand exchange through various degrees of mismatching. We propose that the main roles of hRad51 in homologous recombination is to initiate the homology recognition and strand-exchange steps and those of hRad54 are to promote efficient branch migration, bypass potential mismatches and facilitate long-range strand exchanges through branch migration of Holliday junctions.     

REV7 is required for anaphase-promoting complex-dependent ubiquitination and degradation of translesion DNA polymerase REV1

REV1 is a Y-family polymerase specialized for replicating across DNA lesions at the stalled replication folk. Due to the high error rate of REV1-dependent translesion DNA synthesis (TLS), tight regulation of REV1 activity is essential. Here, we show that human REV1 undergoes proteosomal degradation mediated by the E3 ubiquitin ligase known as anaphase-promoting complex (APC). REV1 associates with APC. Overexpression of APC coactivator CDH1 or CDC20 promotes polyubiquitination and proteosomal degradation of REV1. Surprisingly, polyubiquitination of REV1 also requires REV7, a TLS accessory protein that interacts with REV1 and other TLS polymerases. The N-terminal region of REV1 contains both the APC degron and an additional REV7-binding domain. Depletion of REV7 by RNA interference stabilizes REV1 by preventing polyubiquitination, whereas overexpression of REV7 augments REV1 degradation. Taken together, our findings suggest a role of REV7 in governing REV1 stability and interplay between TLS and APC-dependent proteolysis.     

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance （DDT） has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen （PCNA） by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis （TLS） with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.     

Regulation of Rad51 function by phosphorylation

Rad51 is a key enzyme involved in DNA double-strand break repair by homologous recombination. Here, we show that in response to DNA damage, budding yeast Rad51 is phosphorylated on Ser 192 in a manner that is primarily mediated by the DNA-damage-responsive protein kinase Mec1. We show that mutating Rad51 Ser 192 to Ala or Glu confers hypersensitivity to DNA damage and homologous-recombination defects. Furthermore, biochemical analyses indicate that Ser 192 is required for Rad51 adenosine triphosphate hydrolysis and DNA-binding activity in vitro, whereas mutation of Ser 192 does not interfere with Rad51 multimer formation. These data suggest a model in which Mec1-mediated phosphorylation of Rad51 Ser 192 in response to DNA damage controls Rad51 activity and DNA repair by homologous recombination.