SUMMARY DNA repair and DNA damage tolerance machineries are crucial to overcome the vast array of DNA damage that a cell encounters during its lifetime. In this review, we summarize the current state of knowledge about the eukaryotic DNA damage tolerance pathway translesion synthesis (TLS), a process in which specialized DNA polymerases replicate across from DNA lesions. TLS aids in resistance to DNA damage, presumably by restarting stalled replication forks or filling in gaps that remain in the genome due to the presence of DNA lesions. One consequence of this process is the potential risk of introducing mutations. Given the role of these translesion polymerases in mutagenesis, we discuss the significant regulatory mechanisms that control the five known eukaryotic translesion polymerases: Rev1, Pol ζ, Pol κ, Pol η, and Pol ι.
Background: Translesion synthesis in mammalian cells is achieved by sequential actions of insertion and extension polymerases. Results: We determined the Rev1-Pol -Pol complex structure and verified the binding interface with in vivo studies. Conclusion: Mammalian insertion and extension polymerases could cooperate within a megatranslesion polymerase complex nucleated by Rev1. Significance: The Rev1-Pol interface is a target for developing novel cancer therapeutics.
The DNA mismatch repair machinery is involved in the correction of a wide variety of mutational intermediates. In bacterial cells, homodimers of the MutS protein bind mismatches and MutL homodimers couple mismatch recognition to downstream processing steps [1]. Eukaryotes possess multiple MutS and MutL homologs that form discrete, heterodimeric complexes with specific mismatch recognition and repair properties. In yeast, there are six MutS (Msh1-6p) and four MutL (Mlh1-3p and Pms1p) family members [2] [3]. Heterodimers comprising Msh2p and Msh3p or Msh2p and Msh6p recognize mismatches in nuclear DNA [4] [5] and the subsequent processing steps most often involve a Mlh1p-Pms1P heterodimer [6] [7]. Mlh1p also forms heterodimeric complexes with Mlh2p and Mlh3p [8], and a minor role for Mlh3p in nuclear mismatch repair has been reported [9]. No mismatch repair function has yet been assigned to the fourth yeast MutL homolog, Mlh2p, although mlh2 mutants exhibit weak resistance to some DNA damaging agents [10]. We have used two frameshift reversion assays to examine the roles of the yeast Mlh2 and Mlh3 proteins in vivo. This analysis demonstrates, for the first time, that yeast Mlh2p plays a role in the repair of mutational intermediates, and extends earlier results implicating Mlh3p in mismatch repair.
The use of translesion synthesis (TLS) polymerases to bypass DNA lesions during replication constitutes an important mechanism to restart blocked/stalled DNA replication forks. Because TLS polymerases generally have low fidelity on undamaged DNA, the cell must regulate the interaction of TLS polymerases with damaged versus undamaged DNA to maintain genome integrity. The Saccharomyces cerevisiae checkpoint proteins Ddc1, Rad17, and Mec3 form a clamp-like structure (the 9-1-1 clamp) that has physical similarity to the homotrimeric sliding clamp proliferating cell nuclear antigen, which interacts with and promotes the processivity of the replicative DNA polymerases. In this work, we demonstrate both an in vivo and in vitro physical interaction between the Mec3 and Ddc1 subunits of the 9-1-1 clamp and the Rev7 subunit of the Pol TLS polymerase. In addition, we demonstrate that loss of Mec3, Ddc1, or Rad17 results in a decrease in Pol-dependent spontaneous mutagenesis. These results suggest that, in addition to its checkpoint signaling role, the 9-1-1 clamp may physically regulate Poldependent mutagenesis by controlling the access of Pol to damaged DNA.The major replicative DNA polymerases are high fidelity enzymes that can be blocked by lesion-containing bases on the template strand (1). Although such blockage can potentially prevent completion of genome duplication, cells can bypass lesions by copying information from the undamaged sister chromatid in a strand switching or recombination type of mechanism. Alternatively, a translesion synthesis (TLS) 5 DNA polymerase can be recruited to insert a nucleotide across from the lesion or to extend a lesion-base mispair (reviewed in Refs. 2 and 3). This unique activity of the TLS polymerases has been attributed to the presence of a large catalytic active site that can accommodate structurally deformed bases (4). Although it has been suggested that each type of translesion polymerase may be specialized to bypass a certain lesion (or class of lesions) in a relatively error-free manner, many of the TLS polymerases exhibit astoundingly low fidelity on undamaged DNA in vitro (reviewed in Refs. 2 and 4). To minimize replication errors on undamaged DNA, the in vivo use of translesion polymerases must be restricted so that they are employed only when needed.Saccharomyces cerevisiae contains three translesion polymerases: Pol, Pol, and Rev1. Pol has been primarily characterized with respect to its role in the error-free bypass of UV-induced lesions (5) and this bypass appears to require physical interaction with PCNA (6). Yeast strains lacking REV1 or Pol demonstrate similar phenotypes in response to DNA damage (4), and it is generally assumed that these two polymerases act in the same pathway of mutagenesis (but see Ref. 7). In vitro studies indicate that Rev1 is a G template-specific DNA polymerase (8), but the relevance of this activity to in vivo mutagenesis is unclear (9). Pol, which is comprised of a catalytic subunit encoded by REV3 and a regulatory subunit encoded by REV7...
The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction with the phosphate on the 5′-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed, eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.
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