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Binding studies of human single-stranded DNA binding and repair proteins: Replication protein A and RAD52.

机译：人类单链DNA结合和修复蛋白的结合研究：复制蛋白A和RAD52。

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摘要

Heterotrimeric replication protein A (RPA) is the primary eukaryotic single-stranded DNA (ssDNA) binding protein that plays an essential role in DNA metabolism including replication, recombination and repair. RPA has 6 ssDNA binding domains (DBDs) A-F. It prefers poly-pyrimidine over poly-purine sequences and binds non-canonical DNA structures (G-quadruplexes and triplexes). In order to study the crystal structure of RPA bound to ssDNA, SELEX experiments were performed to identify a specific ssDNA binding sequence for RPA. SELEX experiments with the primary DBDs-A and -B select an 8 nucleotide TC-rich motif, whereas DBDs-C, -D and -E retrieve a 20 nucleotide G-quadruplex forming sequence. Fluorescence polarization binding studies with RPA-AB and RPA-CDE-core show that these DBDs bind pyrimidine- and purine-rich mixed sequences similarly. Interestingly, RPA-DE binds preferentially to the G-quadruplex sequence, a unique preference not observed with other RPA constructs studied. Circular dichroism experiments show RPA-CDE unfolding the G-quadruplex while RPA-DE stabilizes it. Binding of RPA70-C, a construct of RPA that has not been previously purified, to various DNA sequences indicates a universal binder function for this domain. Circular dichroism titration experiments with a longer G-quadruplex forming sequence, containing a TC-rich region 5' to the G-quadruplex, is not unfolded by RPA-CDE-core possibly because RPA70-C binds the TC-rich region while RPA-DE binds the G-quadruplex. Molecular modeling studies of RPA32-D and proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA32-D and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties.;RAD52 and RPA are two key players in the repair of double-stranded DNA breaks (DSBs). The exact mechanism of recruitment of these proteins to damaged DNA is unknown. A small section of this thesis, dedicated to studying the ssDNA binding properties of phosphorylated RPA (pRPA) and RAD52, indicated that pRPA binds with a greater affinity to a mixed ssDNA sequence when compared to RPA, and RAD52 binds ssDNA ends. These results combined with previous studies from the Borgstahl laboratory suggest that phosphorylation of RPA may serve to recruit downstream repair proteins like RAD52 in order to efficiently repair damaged DNA.
机译：异三聚体复制蛋白A（RPA）是主要的真核单链DNA（ssDNA）结合蛋白，在DNA代谢（包括复制，重组和修复）中起重要作用。 RPA具有6个ssDNA结合域（DBD）A-F。与聚嘌呤序列相比，它更喜欢聚嘧啶，并结合非规范的DNA结构（G-四链体和三链体）。为了研究RPA与ssDNA结合的晶体结构，进行了SELEX实验，以鉴定RPA的特定ssDNA结合序列。使用主要DBDs-A和-B进行SELEX实验选择了一个8个核苷酸的富含TC的基序，而DBDs-C，-D和-E检索了一个20个核苷酸的G-四链体形成序列。用RPA-AB和RPA-CDE核心进行的荧光偏振结合研究表明，这些DBD相似地结合了富含嘧啶和嘌呤的混合序列。有趣的是，RPA-DE优先与G-四链体序列结合，这种独特的偏好是在其他RPA构建体中未观察到的。圆二色性实验表明，RPA-CDE使G-四链体展开，而RPA-DE使它稳定。 RPA70-C（一种之前尚未纯化的RPA的构建体）与各种DNA序列的结合表明该结构域具有普遍的结合功能。 RPA-CDE核心无法展开具有较长G-四链体形成序列的循环二色滴定实验，该序列包含一个距G-四链体5'的富含TC的区域，可能是因为RPA70-C结合了TC丰富的区域，而RPA- DE结合G-四链体。 RPA32-D和蛋白质Pot1和Stn1的分子模型研究揭示了蛋白质之间的结构相似性，并阐明了RPA32-D和Stn1的潜在DNA结合位点。这些数据表明RPA的DBD具有不同的ssDNA识别特性。RAD52和RPA是修复双链DNA断裂（DSB）的两个关键因素。这些蛋白质募集到受损的DNA的确切机制尚不清楚。本文的一小部分致力于研究磷酸化RPA（pRPA）和RAD52的ssDNA结合特性，表明与RPA相比，pRPA对混合的ssDNA序列具有更大的亲和力，而RAD52与ssDNA末端结合。这些结果与Borgstahl实验室的先前研究相结合，表明RPA的磷酸化可能有助于募集下游修复蛋白（如RAD52），从而有效修复受损的DNA。

著录项

  • 作者

    Prakash, Aishwarya.;

  • 作者单位

    University of Nebraska Medical Center.;

  • 授予单位 University of Nebraska Medical Center.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 300 p.
  • 总页数 300
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:36:56

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