Michael R. Volkert

Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli.

SummaryCellular activities normally inducible by DNA damage (SOS functions) are expressed, without DNA damage, in recA441 (formerly tif-1) mutants of Escherichia coli at 42° C but not at 30° C. We describe a strain (SC30) that expresses SOS functions (including mutator activity, prophage induction and copious synthesis of recA protein) constitutively at both temperatures. SC30 is one of four stable subclones (SC strains) derived from an unstable recombinant obtained in a conjugation between a recA441 K12 donor and a recA+ B/r-derived recipient. SC30 does not owe its SOS-constitutive phenotype to a mutation in the lexA gene (which codes the repressor of recA and other DNA damage-inducible genes), since it is lexA+. Each of the SC strains expresses SOS functions in a distinctively anomalous way. We show that the genetic basis for the differences in SOS expression among the SC strains is located at or very near the recA locus. We propose that resolution of genetic instability in this region, in the original recombinant, has altered the pattern of expression of SOS functions in the SC strains.

Stress Induction and Mitochondrial Localization of Oxr1 Proteins in Yeast and Humans

ABSTRACT Reactive oxygen species (ROS) are critical molecules produced as a consequence of aerobic respiration. It is essential for cells to control the production and activity of such molecules in order to protect the genome and regulate cellular processes such as stress response and apoptosis. Mitochondria are the major source of ROS within the cell, and as a result, numerous proteins have evolved to prevent or repair oxidative damage in this organelle. The recently discovered OXR1 gene family represents a set of conserved eukaryotic genes. Previous studies of the yeast OXR1 gene indicate that it functions to protect cells from oxidative damage. In this report, we show that human and yeast OXR1 genes are induced by heat and oxidative stress and that their proteins localize to the mitochondria and function to protect against oxidative damage. We also demonstrate that mitochondrial localization is required for Oxr1 protein to prevent oxidative damage.

The Single-Strand DNA Binding Activity of Human PC4 Prevents Mutagenesis and Killing by Oxidative DNA Damage

ABSTRACT Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub1Δ mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

The Escherichia coli Methyl-Directed Mismatch Repair System Repairs Base Pairs Containing Oxidative Lesions

A major role of the methyl-directed mismatch repair (MMR) system of Escherichia coli is to repair postreplicative errors. In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis. When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically. In addition, we show that a dam mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by mutations that inactivate MMR. In a dam mutant, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted to single- and double-strand DNA breaks. Thus, base pairs containing oxidized bases will be converted to strand breaks if they are repaired by MMR. This is demonstrated by the increased peroxide sensitivity of a dam mutant and the finding that the sensitivity can be suppressed by mutations inactivating MMR. We demonstrate further that this repair activity results from MMR recognition of base pairs containing 8-oxoguanine (8-oxoG) based on the finding that overexpression of the MutM oxidative repair protein, which repairs 8-oxoG, can suppress the mutH-dependent increase in transversion mutations. These findings demonstrate that MMR has the ability to prevent oxidative mutagenesis either by removing 8-oxoG directly or by removing adenine misincorporated opposite 8-oxoG or both.

Regulatory Responses of the Adaptive Response to Alkylation Damage: a Simple Regulon with Complex Regulatory Features

Alkylation damage to DNA occurs when cells encounter alkylating agents in the environment or when cellular metabolism produces active alkylators. To cope with DNA alkylation, cells have evolved genes that encode proteins with alkylation-specific DNA repair activities. In Escherichia coli , the main

Deregulation of DNA Damage Signal Transduction by Herpesvirus Latency-Associated M2

ABSTRACT Infected cells recognize viral replication as a DNA damage stress and elicit a DNA damage response that ultimately induces apoptosis as part of host immune surveillance. Here, we demonstrate a novel mechanism where the murine gamma herpesvirus 68 (γHV68) latency-associated, anti-interferon M2 protein inhibits DNA damage-induced apoptosis by interacting with the DDB1/COP9/cullin repair complex and the ATM DNA damage signal transducer. M2 expression constitutively induced DDB1 nuclear localization and ATM kinase activation in the absence of DNA damage. Activated ATM subsequently induced Chk activation and p53 phosphorylation and stabilization without eliciting H2AX phosphorylation and MRN recruitment to foci upon DNA damage. Consequently, M2 expression inhibited DNA repair, rendered cells resistant to DNA damage-induced apoptosis, and induced a G1 cell cycle arrest. Our results suggest that γHV68 M2 blocks apoptosis-mediated intracellular innate immunity, which might ultimately contribute to its role in latent infection.

RNA polymerase alpha subunit binding site in positively controlled promoters: a new model for RNA polymerase-promoter interaction and transcriptional activation in the Escherichia coli ada and aidB genes.

The ada and aidB genes are part of the adaptive response to DNA methylation damage in Escherichia coli. Transcription of the ada and the aidB genes is triggered by binding of the methylated Ada protein (meAda) to a specific sequence located 40–60 base pairs upstream of the transcriptional start, which is internal to an A/T‐rich region. In this report we demonstrate that the Ada binding site is also a binding site for RNA polymerase. RNA polymerase is able to bind the −40 to −60 region of the ada and the aidB promoters in the absence of meAda, and its binding is mediated by the alpha subunit. This region resembles the UP element of the rrnB P1 promoter in location, sequence and mechanism of interaction with RNA polymerase. We discuss the function of UP‐like elements in positively controlled promoters and provide evidence that Ada does not act by enhancing RNA polymerase binding affinity to the promoter region. Instead, Ada stimulates transcription by modifying the nature of the RNA polymerase‐promoter interaction, allowing RNA polymerase to recognize the core promoter −35 and −10 elements in addition to the UP‐like element.

The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins.

BackgroundThe NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1.ResultsWe find that NCOA7, like OXR1 can suppress the oxidative mutator phenotype when expressed in an E. coli strain that exhibits an oxidation specific mutator phenotype. Moreover, NCOA7s oxidation resistance function requires expression of only its carboxyl-terminal domain and is similar in this regard to OXR1. We find that, in human cells, NCOA7 is constitutively expressed and is not induced by oxidative stress and appears to localize to the nucleus following estradiol stimulation. These properties of NCOA7 are in striking contrast to those of OXR1, which is induced by oxidative stress, localizes to mitochondria, and appears to be excluded, or largely absent from nuclei.ConclusionNCOA7 most likely arose from duplication. Like its homologue, OXR1, it is capable of reducing the DNA damaging effects of reactive oxygen species when expressed in bacteria, indicating the protein has an activity that can contribute to oxidation resistance. Unlike OXR1, it appears to localize to nuclei and interacts with the estrogen receptor. This raises the possibility that NCOA7 encodes the nuclear counterpart of the mitochondrial OXR1 protein and in mammalian cells it may reduce the oxidative by-products of estrogen metabolite-mediated DNA damage.

Ada Protein-RNA Polymerase ς Subunit Interaction and α Subunit-Promoter DNA Interaction Are Necessary at Different Steps in Transcription Initiation at the Escherichia coli ada andaidB Promoters

The methylated form of the Ada protein (meAda) binds the ada andaidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of theEscherichia coli ada and aidB genes. This region is also a binding site for the α subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter. In this report, we show that deletion of the C-terminal domain of the α subunit severely decreasesmeAda-independent binding of RNA polymerase toada and aidB, affecting transcription initiation at these promoters. We provide evidence thatmeAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase ς70 subunit (amino acids 574–613). Several negatively charged residues in the ς70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect onmeAda-dependent transcription. Based on these observations, we propose that the role of the α subunit atada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on meAda-ς70 interaction.

A NEW CLASSIFICATION OF PATHWAYS REPAIRING PYRIMIDINE DIMER DAMAGE IN DNA

ABSTRACT Pathways of repair of pyrimidine-dimer damage are classified as intrareplicational if they operate on incompletely replicated segments of a chromosome and extrareplicational if they operate on unreplicated and completely replicated segments. Intrareplicational repair is divided into four pathways: 1. Transdimer synthesis (formerly SOS repair, mutation-prone repair and induced repair) 2. & 3. RecBC and alternate RecF pathways of breakage-reunion gap-filling (formerly postreplication repair and recombination repair) 4. Copy-choice excision (a new proposal). Extrareplicational repair is divided into three pathways: 1. Short patch excision which operates on unreplicated and completely replicated chromosome segments 2. Long patch excision which may operate only on completely replicated segments and 3. Incision-promoted-recombinational excision which may be the mechanism of long patch excision. Reliance of these pathways on particular genes is discussed as is their possible evolutionary significance.