DNA Replication, Repair, and Mutagenesis In Eukaryotic And Prokaryotic Cells

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Scientists in the Section on DNA Replication, Repair and Mutagenesis (SDRRM) study the mechanisms by which mutations are introduced into DNA. These studies have traditionally spanned the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes and involve collaborations with scientists around the world. The Escherichia coli dnaE gene encodes the alpha-catalytic subunit (pol III alpha) of DNA polymerase III, the cell's main replicase. Like all high-fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so-called steric gate residue in the active site of the polymerase that physically clashes with the 2'-OH of an incoming ribonucleotide. Our structural modeling data suggests that H760 is the steric gate residue in E.coli pol III alpha. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (alpha, theta, and epsilon). Of the possible 38 mutants, only 9 were successfully sub-cloned: 3 with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid-encoded alleles, S759C, S759N and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature. In a collaboration with Myron Goodman (University of Southern California), we explored the role of the beta-sliding processivity clamp on the efficiency of E.coli DNA polymerase V (pol V), catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of beta. The results suggest that the beta-sliding clamp may have two stabilizing roles: its canonical role in tethering the polymerase at a primer-3'-terminus, and a possible second role in inhibiting pol Vs ATPase, so as to reduce the rate of mutasome-DNA dissociation. We extended our prokaryotic Ribonucleotide Excision Repair (RER) studies in a collaboration with Anders Clausen (University of Gothenburg, Sweden). Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that were deficient in ribonucleotide excision repair (delta rnhB), deficient in pol IV DNA polymerase, constitutively express all SOS-regulated genes lexA(Def) and constitutively activated RecA* (recA730). The strains also harbor two steric gate variants of E. coli pol V (Y11A or F10L), or a homolog of pol V, (pol VR391-Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol VR391-Y13A variants, with a preference to the lagging strand. In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference was observed. Sharp transitions in strand specificity were observed at the replication origin (oriC), while a gradient was observed at the termination region. To activate RecA* in a recA+ strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the pol V-Y11A steric gate variant exhibited a lagging strand preference. Our data are therefore consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response. When subcloned into low-copy-number expression vectors, rumAB, encoding pol VR391 (RumA'2 B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polVR391 is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis-acting factors that suppress the expression and/or the activity of polVR391. Indeed, we recently discovered that SetRR391, an ortholog of lambda cI repressor, is a transcriptional repressor of rumAB. Our studies revealed that CroSR391, an ortholog of lambda Cro, also serves as a potent transcriptional repressor of rumAB. Levels of RumA are dependent upon an interplay between SetRR391 and CroSR391, with the greatest reduction of RumA protein levels observed in the absence of SetRR391 and the presence of CroSR391. Under these conditions, CroSR391 completely abolishes the high levels of mutagenesis promoted by polVR391 expressed from low-copy-number plasmids. Furthermore, deletion of croSR391 on the native R391 results in a dramatic increase in mutagenesis, indicating that CroSR391 plays a major role in suppressing polVR391 mutagenesis in vivo. Inactivating mutations in CroSR391 therefore have the distinct possibility of increasing cellular mutagenesis that could lead to the evolution of antibiotic resistance of pathogenic bacteria harboring R391. In another scientific collaboration with Irina Bezsonova (University of Connecticut Health Center), we investigated the regulation of human DNA polymerase iota (Pol iota), which was discovered by scientists in the SDRRM twenty-two years ago. Like E.coli Pol V, Pol iota belongs to the Y-family of DNA polymerases that are involved in DNA damage tolerance through their role in translesion DNA synthesis. Reversible protein ubiquitination is an essential signaling mechanism within eukaryotes. Deubiquitinating enzymes are critical to this process, as they mediate removal of ubiquitin from substrate proteins. Ubiquitin-specific protease 7 (USP7) is a prominent deubiquitinating enzyme, with an extensive network of interacting partners and established roles in cell cycle activation, immune responses and DNA replication. Characterized USP7 substrates primarily interact with one of two major binding sites outside the catalytic domain. These are located on the USP7 N-terminal TRAF-like (TRAF) domain and the first and second UBL domains (UBL1-2) within the C-terminal tail. Our studies revealed that Pol iota is a novel USP7 substrate that interacts with both TRAF and UBL1-2. Through the use of biophysical approaches and mutational analysis, we characterized both interfaces and demonstrated that bipartite binding to both USP7 domains is required for efficient Pol iota de-ubiquitination. Together, our data established a new bipartite mode of USP7 substrate binding.