Pivotal role of the beta-clamp in translesion DNA synthesis and mutagenesis in E. coli cells

The genetic information is continuously subjected to the attack by endogenous and exogenous chemical and physical carcinogens that damage the DNA template, thus compromising its biochemical functions. Despite the multiple and efficient DNA repair systems that have evolved to cope with the large variety of damages, some lesions may persist and, as a consequence, interfere with DNA replication. By essence, the damaged-DNA replication process (hereafter termed translesion synthesis or TLS) is a major source of point mutations and is therefore deeply involved in the onset of human diseases such as cancer. Recent identification of numerous DNA polymerases involved in TLS has shed new light onto the molecular mechanisms of mutagenesis. Here, we show that in vivo, both error-free and mutagenic bypass activities of the three DNA polymerases known to be involved in TLS in Escherichia coli (PolII, PolIV and PolV) strictly depend upon the integrity of small peptidic sequences identified as their beta-clamp binding motif. Thus, in addition to its crucial role as the processivity factor of the PolIII replicase, the beta-clamp plays a pivotal role during the TLS process.     

DNA polymerase I-mediated translesion synthesis in RecA-independent DNA interstrand cross-link repair in E. coli

DNA interstrand cross-links (ICLs) are mainly repaired by the combined action of nucleotide excision repair and homologous recombination in E. coli. Genetic data also suggest the existence of a nucleotide excision repair-dependent, homologous recombination-independent ICL repair pathway. The involvement of translesion synthesis in this pathway has been postulated; however, the molecular mechanism of this pathway is not understood. To examine the role of translesion synthesis in ICL repair, we generated a defined substrate with a single psoralen ICL that mimics a postincision structure generated by nucleotide excision repair. We demonstrated that the Klenow fragment (DNA polymerase I) performs translesion synthesis on this model substrate. This in vitro translesion synthesis assay will help in understanding the basic mechanism of a postincision translesion synthesis process in ICL repair.     

Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition

Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD' and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD' and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD' and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus.     

Escherichia coli UmuC active site mutants: Effects on translesion DNA synthesis, mutagenesis and cell survival

Escherichia coli polymerase V (pol V/UmuD(2)'C) is a low-fidelity DNA polymerase that has recently been shown to avidly incorporate ribonucleotides (rNTPs) into undamaged DNA. The fidelity and sugar selectivity of pol V can be modified by missense mutations around the "steric gate" of UmuC. Here, we analyze the ability of three steric gate mutants of UmuC to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro, and to promote UV-induced mutagenesis and cell survival in vivo. The pol V (UmuC_F10L) mutant discriminates against rNTP and incorrect dNTP incorporation much better than wild-type pol V and although exhibiting a reduced ability to bypass a CPD in vitro, does so with high-fidelity and consequently produces minimal UV-induced mutagenesis in vivo. In contrast, pol V (UmuC_Y11A) readily misincorporates both rNTPs and dNTPs during efficient TLS of the CPD in vitro. However, cells expressing umuD'C(Y11A) were considerably more UV-sensitive and exhibited lower levels of UV-induced mutagenesis than cells expressing wild-type umuD'C or umuD'C(Y11F). We propose that the increased UV-sensitivity and reduced UV-mutability of umuD'C(Y11A) is due to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than an inability to traverse UV-induced lesions.     

Evidence for in vitro translesion DNA synthesis past a site-specific aminofluorene adduct

The ability of Escherichia coli DNA polymerase I and T7 DNA polymerase to bypass bulky C-8 guanyl-2-aminofluorene adducts in DNA was studied by in vitro DNA synthesis reactions on a site-specific aminofluorene-modified M13mp9 template. This site-specifically modified DNA was prepared by ligating an oligonucleotide containing a single aminofluorene adduct into a gapped heteroduplex of M13mp9 DNA (Johnson, D. L., Reid, T. M., Lee, M.-S., King, C. M., and Romano, L. J. (1986) Biochemistry 25, 449-456). The resulting covalently closed duplex DNA molecule was then cleaved with a restriction endonuclease, denatured, and annealed to a primer on the 3' side of the adduct to form a template specifically designed to study bypass. In this system, any synthesis that was not blocked by the bulky aminofluorene adduct would proceed to the 5' terminus of the single-stranded template, while synthesis interrupted by the adduct would terminate at or near the adduct location. We have measured DNA synthesis on this template and find that the amount of radiolabeled nucleotide incorporated by either E. coli DNA polymerase I (large fragment) or T7 DNA polymerase was much greater than would be predicted if the aminofluorene adduct were an absolute block to DNA synthesis. Furthermore, the products of similar reactions electrophoresed on polyacrylamide gels showed conclusively that the majority of the DNA synthesized by either the T7 DNA polymerase or E. coli DNA polymerase I bypassed the aminofluorene lesion. Substitution of Mn2+ for Mg2+ as the divalent cation resulted in even higher levels of translesion synthesis.     

Error-free recombinational repair predominates over mutagenic translesion replication in E. coli

Tolerance mechanisms are important in the ability of cells to cope with DNA damage. In E. coli, the two main damage tolerance mechanisms are recombinational repair (RR) and translesion replication (TLR). Here we show that RR effectively repairs gaps opposite DNA lesions. When both mechanisms are functional, RR predominates over TLR, being responsible for 86% of the repair events. This predominance of RR is determined by the high concentration of RecA present under SOS conditions, which causes a differential inhibition of TLR. Further inhibition of TLR is caused by the RecA-catalyzed strand exchange reaction of RR. This molecular hierarchy in the tolerance of DNA lesions ensures that the nonmutagenic RR predominates over the mutagenic TLR, thereby contributing to genetic stability.     

Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein

Summary We have used a sensitive gel electrophoresis assay to detect the products of Escherichia coli RecA protein catalysed strand exchange reactions between gapped and duplex DNA molecules. We identify structures that correspond to joint molecules formed by homologous pairing, and show that joint molecules are converted by RecA protein into heteroduplex monomers by reciprocal strand exchanges. However, strand exchanges only occur when there is a 3-terminus complementary to the single stranded DNA in the gap. In the absence of a complementary free end, the two DNA molecules pair and short heteroduplex regions are formed by localised interwinding.     

Site-specific mutagenesis using asymmetric polymerase chain reaction and a single mutant primer.

A method is described for preparing site-specific mutants using a polymerase chain reaction (PCR) based protocol. The protocol requires a single mutant primer, and has been used to introduce mutations into DNA fragments ranging in size from 200 bp to 1569 bp in length in the GM-CSF, beta-actin, human growth hormone and erythropoietin genes. Sequence analysis of PCR derived mutant fragments shows an error rate of less than one bp change per 1500 bp incorporated. Single base pair mutations have been introduced into these genes which create unique restriction sites. We demonstrate that these mutant templates may be used for competitive PCR to quantitate mRNA and DNA. This method thus offers a rapid means for producing competitive templates for use in quantitative PCR.     

Site-specific dissection of E. coli chromosome by lambda terminase.

We have succeeded the targeted cleavage of chromosomes by lambda terminase that introduces double-strand cleavages in DNA recognizing the lambda cos sequence. When chromosomal DNAs of various Escherichia coli K-12 strains were subjected to terminase digestion, all were found to contain two common cleavage sites. Therefore, DNAs from lambda lysogens in which lambda DNA was inserted at different chromosomal sites were specifically cleaved at one more additional site. The two sites, termed ecos1 and ecos2, were mapped at approximately 35.1' and 12.7' of E. coli genetic map. The ecos1 and ecos2 sites were included in qin and qsr' regions, respectively. Therefore, the cleavage sites were associated with cryptic prophages. Sequences at the ecos1 and ecos2 sites showed 98% homology to the lambda cos sequence, indicating high fidelity of sequence recognition by the terminase. Since the strategy for integration of a DNA segment into chromosomal DNA through homologous recombination has been established, the dissection method that uses lambda terminase should be applicable for gene mapping as well as construction of macrophysical maps of larger genomes.     

Site-specific cleavage of DNA by E. coli DNA gyrase.

E. coli DNA gyrase, which catalyzes the supercoiling of DNA, cleaves DNA site-specifically when oxolinic acid and sodium dodecylsulfate are added to the reaction. We studied the structure of the gyrasecleaved DNA because of its implications for the reaction mechanism and biological role of gyrase. Gyrase made a staggered cut, creating DNA termini with a free 3' hydroxyl and a 5' extension that provided a template primer for DNA polymerase. The cleaved DNA was resistant to labeling with T4 polynucleotide kinase even after treatment with proteinase K. Thus the denatured enzyme that remains attached to cleaved DNA is covalently bonded to both 5' terminal extensions. The 5' extensions of many gyrase cleavage fragments from phi X174, SV40 and Col E1 DNA were partially sequenced using repair with E. coli DNA polymerase I. No unique sequence existed within the cohesive ends, but G was the predominant first base incorporated by DNA polymerase I. The cohesive and sequences of four gyrase sites were determined, and they demonstrated a four base 5' extension. The dinucleotide TG, straddling the gyrase cut on one DNA strand, provided the only common bases within a 100 bp region surrounding the cleavage sites. Analysis of other cleavage fragments showed that cutting between a TG doublet is common to most, or all, gyrase cleavages. Other bases common to some of the sequenced sites were clustered nonrandomly around the TG doublet, and may be variable components of the cleavage sequence. This diverse recognition sequence with common elements is a pattern shared with several other specific nucleic acid-protein interactions.     

sRNASVM——基于SVM方法构建大肠杆菌sRNA预测模型

在理解细菌与环境的相互作用方面,细菌sRNA的识别发挥重要作用。文章介绍了一个通过增加训练集中实验证实的sRNA来构建细菌sRNA预测模型的策略,并以大肠杆菌K-12的sRNA预测为例来说明策略的可行性。结果表明,按此策略构建的模型sRNASVM的10倍交叉检验精度达到92.45%,高于目前文献中报道的精度。因此,构建的这一模型将为实验发现sRNA提供较好的生物信息学支持。有关模型和详细结果可以从网站http://ccb.bmi.ac.cn/srnasvm/下载。     

Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro.

The gapped duplex DNA approach to oligonucleotide-directed construction of mutations (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) has been developed further. A procedure is described that makes in vitro DNA polymerase/DNA ligase reactions dispensable. Direct transfection of host bacteria with gdDNA molecules of recombinant phage M13 plus mutagenic oligonucleotide results in marker yields in excess of 50% (gap size 1640 nucleotides). An important feature incorporated into the mutagenic oligonucleotide is the presence of one or two internucleotidic phosphorothioate linkages immediately adjacent to the 5'-terminus. Automated preparation and biochemical properties of such compounds are described as well as their performance in oligonucleotide-directed mutagenesis. A systematic study of the following parameters influencing marker yield is reported: Gap size, length of oligonucleotide, chemical nature of oligonucleotide termini and heatshock temperature during transformation.     

Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli

The site-specific modification of proteins is expected to be an important capability for the synthesis of bioconjugates in the future. However, the traditional repertoire of reactions available for the direct modification of proteins suffers from lack of specificity, necessitating costly downstream processing to isolate the specific species of interest. (1) Here, we use a well-established, glycan-specific chemistry to PEGylate model glycoproteins, each containing a unique reactive GalNAc attached to a specifically engineered threonine residue. By engineering E. coli to execute the initial steps of human, mucin-type O-glycosylation, we were able to obtain homogeneous site-specifically modified glycoproteins with fully human glycan linkages. Two mucin-based reporters as well as several fusion proteins containing eight-amino-acid GalNAc-T recognition sequences were glycosylated in this engineered glycocompetent strain of E. coli. The use of one sequence in particular, PPPTSGPT, resulted in site-specific glycan occupancy of approximately 69% at the engineered threonine. The GalNAc present on the purified glycoprotein was oxidized by galactose oxidase and then coupled to hydroxylamine functionalized 20 kDa PEG in the presence of aniline. The glycoprotein could be converted to the PEGylated product at approximately 85% yield and >98% purity as determined by comparison to the products of control reactions.     

Enhancement by cysteamine of N-methyl-N-nitrosourea mutagenesis in E. coli

Cysteamine (MEA) is comutagenic to methylnitrosourea (MNU) in E. coli AB 1157 but not in the nonadaptable mutant derivative ada-6 of that strain. The comutagenic action of MEA was eliminated by cysteine at low concentrations, which also lowered mutation frequencies in AB1157 but not in ada-6. In model experiments it was shown that cysteine counteracted the inhibition by MEA of beta-galactosidase induction in both bacterium strains. The comutagenic action of MEA is interpreted as being due to an inhibition of induction of methyltransferase during treatment with MNU.     

Site-directed mutagenesis of aspartate aminotransferase from E. coli

The gene for aspartate aminotransferase from E. coli (aspC) was subcloned into M13 phage and sequenced using the Sanger dideoxy method with synthetic oligonucleotide primers. A mutant gene was constructed using site-directed mutagenesis techniques in which the codon for the lysine that forms the Schiffs base with pyridoxal phosphate was replaced with one coding for alanine. The mutant gene was expressed under control of the Tac promoter to overproduce a mutant protein lacking enzymatic activity.     

DNA polymerases provide a canon of strategies for translesion synthesis past oxidatively generated lesions

Deducing the structure of the DNA double helix in 1953 implied the mode of its replication: Watson-Crick (WC) base pairing might instruct an enzyme, now known as the DNA polymerase, during the synthesis of a daughter stand complementary to a single strand of the parental double helix. What has become increasingly clear in the last 60 years, however, is that adducted and oxidatively generated DNA bases are ubiquitous in physiological DNA, and all organisms conserve multiple DNA polymerases specialized for DNA synthesis opposite these damaged templates. Here, we review recent crystal structures depicting replicative and bypass DNA polymerases encountering two typical lesions arising from the oxidation of DNA: abasic sites, which block the replication fork, and the miscoding premutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG).