Creation and Repair of Postreplicative DNA Gaps

复制后 DNA 缺口的产生和修复

• 批准号: 10614989
• 负责人: Michael M. Cox
• 金额: $ 121.89万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10614989
• 关键词: ATP phosphohydrolase; Address; Affect; Aging; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Binding Proteins; Biochemical; Biochemistry; Biological; Biophysics; Bypass; Cells; Collaborations; Complex; DNA; DNA Damage; DNA Polymerase II; DNA Repair; DNA Repeat Expansion; DNA Sequence Rearrangement; DNA biosynthesis; DNA lesion; DNA metabolism; DNA polymerase V; DNA replication fork; DNA-Directed DNA Polymerase; Development; Disease; ERCC3 gene; Ensure; Enzymes; Escherichia coli; Eukaryota; Event; Evolution; Expanded DNA Repeat; Frequencies; Gene Rearrangement; Generation Gaps; Genetic; Genetic Recombination; Genome Stability; Genomic DNA; Genomic Instability; Genomics; Goals; Growth; Homologous Gene; Human; Investigation; Left; Lesion; Link; Maintenance; Malignant Neoplasms; Mammals; Mediating; Methodology; Methods; Molecular; Molecular Biology; Mutagenesis; Organism; Pathogenicity; Pathway interactions; Play; Polymerase; Process; Protein Family; Proteins; Research; Resistance development; Role; SOS Response; Site; Source; System; Work; Yeasts; biological adaptation to stress; daughter strand; defined contribution; fitness; in vivo; multidisciplinary; novel; pathogen; pathogenic bacteria; programs; recombinational repair; recruit; repaired; single molecule; skip lesion; tumor; tumor growth

PROJECT SUMMARY/ABSTRACT When a replication fork encounters a DNA lesion in a template strand, replication gives way to DNA repair and recombination. These encounters define an interface in DNA metabolism that gives rise to much of the DNA rearrangement, repeat expansion, and mutagenesis that defines genome instability. This is ultimately manifested in tumor evolution in eukaryotes and the development of antibiotic resistance and increased pathogenicity in bacteria. Recent investigation of events that occur at the fork have largely overlooked an important genomic venue for repair – lesions left behind the fork in postreplicative gaps. The existence of these gaps has been appreciated for over 5 decades, but progress has been limited by methodology that has been inadequate to properly explore their general importance and repair. These gaps are primary substrates for DNA synthesis by translesion DNA polymerases, recombinational DNA repair, and replicational template switching, all processes linked with genomic instability. This proposal frames a multidisciplinary effort to explore how postreplicative gaps are generated, how often they are formed, what circumstances trigger formation, what occurs within them, and how the various paths of gap repair are prioritized and governed. The work is an outgrowth of advances in understanding three enigmatic protein activities, RarA, Uup, and RadD. RarA, an AAA+ ATPase that functions at the replisome to generate postreplicative gaps on the lagging strand, is one key. To tackle this problem, we bring together world-class expertise in biochemistry, genetics, molecular biology, and biophysics. We will develop new methods, including novel single-molecule approaches towards detecting and quantifying gaps, and characterizing the proteins acting on them. While driven by our mechanistic questions, these methods will broadly benefit research in genomic maintenance. The five specific aims constitute a systematic attack on the problem. The mechanism of RarA protein is the focus of Aim 1. Aim 2 provides the first effort to quantify gap formation in vivo and determine the factors that trigger gap formation. Aim 3 explores the functions of Uup and RadD, two enzymes that suppress template switching within gaps. Aim 4 explores how various pathways of gap repair are organized and governed. The proposal is completed with Aim 5, an exploration of translesion DNA polymerases acting within gaps, a key source of genomic mutagenesis.

项目摘要/摘要 当复制叉遇到模板链中的DNA病变时，复制让位于DNA修复 和重组。 DNA重新排列，重复膨胀和定义基因组不稳定性的Mutajenesis 体现在真核生物的肿瘤演化和抗生素耐药性的发展中，并增加 细菌的致病性。 维修的重要基因组场所 - 叉上叉的后叉。 差距已受到超过5年的赞赏，但是进步受到了一直以来的方法的限制 不足以适当地进行这些差距。 通过翻译DNA聚合酶，重组DNA修复和复制模板的DNA合成 切换，与基因组不稳定性相关的所有过程。 该提案构成了多学科的努力，以探讨如何产生后差距 它们是形成的，在情况下触发形成，其中发生了什么，以及如何 差距维修优先考虑，并管理着工作的进步 神秘的蛋白质活性，rara，uup和radd。 在滞后链上生成后上的间隙是一个关键。 为了解决这个问题，我们将生物化学，遗传学，分子的世界一流专业知识汇总在一起 生物学和生物物理学。 检测和量化差距，并表征作用时作用的蛋白质。 机械性问题，这些方法将广泛受益于基因组维持的研究。 五个特定的目标是对问题的系统攻击。 目标1。AIM2提供了第一个努力，以量化体内差距形成并​​确定因素的因素 触发缝隙形成3。 在间隙内进行切换 提案是通过AIM 5完成的，这是对缝隙内的翻译DNA聚合酶的探索，一个钥匙 基因组诱变的来源。

项目成果

RadD is a RecA-dependent accessory protein that accelerates DNA strand exchange.

• DOI: 10.1093/nar/gkac041
• 发表时间: 2022-02-28
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Bonde NJ;Romero ZJ;Chitteni-Pattu S;Cox MM
• 通讯作者: Cox MM

The Escherichia coli serS gene promoter region overlaps with the rarA gene.

• DOI: 10.1371/journal.pone.0260282
• 发表时间: 2022
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Jain K;Stanage TH;Wood EA;Cox MM
• 通讯作者: Cox MM

RecA-independent recombination: Dependence on the Escherichia coli RarA protein.

• DOI: 10.1111/mmi.14655
• 发表时间: 2021-06
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Jain K;Wood EA;Romero ZJ;Cox MM
• 通讯作者: Cox MM

Genomic landscape of single-stranded DNA gapped intermediates in Escherichia coli.

• DOI: 10.1093/nar/gkab1269
• 发表时间: 2022-01-25
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Pham P;Shao Y;Cox MM;Goodman MF
• 通讯作者: Goodman MF

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.

• DOI: 10.1093/nar/gkad186
• 发表时间: 2023-04-24
• 期刊: Nucleic acids research
• 影响因子: 14.9