MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutL␣ (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on the integrity of a PMS2 DQHA(X) 2 E(X) 4 E motif (Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutL␣, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutL␣ is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutS␣, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X) 2 E(X) 4 E motif abolishes yMutL␣ endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutL␣ ATPase activity or the ability of the protein to support assembly of the yMutL␣⅐yMutS␣⅐heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutL␣ incision to the discontinuous strand of a nicked heteroduplex.
Human DNA polymerase theta (pol θ or POLQ) is a proofreading-deficient family A enzyme implicated in translesion synthesis (TLS) and perhaps in somatic hypermutation (SHM) of immunoglobulin genes. These proposed functions and kinetic studies imply that pol θ may synthesize DNA with low fidelity. Here, we show that when copying undamaged DNA, pol θ generates single base errors at rates 10- to more than 100-fold higher than for other family A members. Pol θ adds single nucleotides to homopolymeric runs at particularly high rates, exceeding 1% in certain sequence contexts, and generates single base substitutions at an average rate of 2.4 × 10−3, comparable to inaccurate family Y human pol κ (5.8 × 10−3) also implicated in TLS. Like pol κ, pol θ is processive, implying that it may be tightly regulated to avoid deleterious mutagenesis. Pol θ also generates certain base substitutions at high rates within sequence contexts similar to those inferred to be copied by pol θ during SHM of immunoglobulin genes in mice. Thus, pol θ is an exception among family A polymerases, and its low fidelity is consistent with its proposed roles in TLS and SHM.
Human DNA polymerase ν is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol ν has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol ν lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol ν DNA synthesis fidelity as a clue to its function, here we describe human pol ν error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ α-complementation sequence in M13mp2 DNA. Pol ν copies this DNA with average singlebase insertion and deletion error rates of 7 × 10 −5 and 16 × 10 −5 , respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol γ, and much higher than that of Y family TLS polymerases. In contrast, the average single base substitution error rate of human pol ν is 3.5 × 10 −3 , which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol ν reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol ν error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol ν that could contribute to this unique error signature.
Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2 0 ,3 0 -cyclic phosphates at ribonucleotide sites. Here, we provide genetic and biochemical evidence that DNA double-strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. We show that RNase H2-deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1-induced damage at ribonuclelotide sites. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1-induced DNA nicks at ribonucleotide sites. Analysis of Top1-linked DNA from pull-down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2-deficient yeast cells, supporting this model. Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.