HELICASE ACTIVITY AND ITS ROLE IN TELOMERE AND TELOMERASE REGULATION

解旋酶活性及其在端粒和端粒酶调节中的作用

• 批准号: 9892425
• 负责人: Roberto Galletto
• 金额: $ 5.93万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9892425
• 关键词: Affect; Binding Proteins; DNA; DNA Binding; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Equilibrium; Family; G-Quartets; Homologous Gene; Mutation; Physiological; Property; Proteins; Regulation; Role; SS DNA BP; Site; Structure; Telomerase; Testing; ds-DNA; helicase; melting; monomer; telomere

Abstract/Summary In this proposal we focus on the Pif1 sub-family of DNA helicases and test the hypothesis that one of their functions is to facilitate DNA replication across naturally occurring barriers. We showed that DNA-bound Rap1 is a barrier to the strand-displacement DNA synthesis activity of DNA polymerase  and that Pif1 displaces Rap1, allowing DNA replication across the barrier. We hypothesize that proteins bound to the DNA, at telomeres and non-telomeric sites, are displaced by Pif1 to facilitate DNA replication, and that this activity is fundamental and conserved among Pif1 homologues. G-quadruplexes and DNA hairpins can also be obstacles to DNA replication. Pif1 is thought to facilitate replication-fork progression by unwinding G-quadruplexes. However, little is known about this activity of Pif1 in the presence of single-stranded DNA binding proteins (SSBs). We hypothesize that the activity of Pif1 is needed to facilitate DNA replication across G- quadruplexes when SSBs cannot effectively melt the DNA secondary structure. Furthermore, we hypothesize that Pif1 stimulates DNA synthesis by DNA polymerase  across DNA hairpins. We showed that unwinding of dsDNA by a Pif1 monomer is balanced by a Pif1-dependent DNA rewinding activity. We hypothesize that under physiological conditions rewinding is inhibited by concomitant synthesis of DNA by polymerases. Mutations of Pif1 that affect the balance between unwinding and rewinding will allow us to determine the origin of this newly discovered property. To test these hypotheses, we propose three Aims: 1) determine whether protein obstacles that impede DNA replication are displaced by Pif1; 2) determine whether DNA secondary structures are unwound by Pif1 to facilitate DNA replication; 3) determine the mechanism underlying the Pif1-dependent DNA rewinding activity.

抽象/总结

项目成果

A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End.

• DOI: 10.1016/j.jmb.2016.02.017
• 发表时间: 2016-03-27
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Singh SP;Koc KN;Stodola JL;Galletto R
• 通讯作者: Galletto R

The signature motif of the Saccharomyces cerevisiae Pif1 DNA helicase is essential in vivo for mitochondrial and nuclear functions and in vitro for ATPase activity.

• DOI: 10.1093/nar/gky655
• 发表时间: 2018-09-19
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Geronimo CL;Singh SP;Galletto R;Zakian VA
• 通讯作者: Zakian VA

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5'-flaps.

• DOI: 10.1093/nar/gkv260
• 发表时间: 2015-04-30
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Koc KN;Stodola JL;Burgers PM;Galletto R
• 通讯作者: Galletto R

Translocation of Saccharomyces cerevisiae Pif1 helicase monomers on single-stranded DNA.

• DOI: 10.1093/nar/gkt117
• 发表时间: 2013-04
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Galletto R;Tomko EJ
• 通讯作者: Tomko EJ

The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes.

酵母RAP1的DNA结合结构域与多种结合模式中的双链DNA相互作用。

• DOI: 10.1021/bi501049b
• 发表时间: 2014-12-09
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Feldmann, Erik A.;Galletto, Roberto
• 通讯作者: Galletto, Roberto