Journal article
Measuring the contributions of helicases to break-induced replication
Methods in enzymology, Vol.672, pp.339-368
01/01/2022
DOI: 10.1016/bs.mie.2022.02.025
Abstract
Break-Induced Replication (BIR) is a homologous recombination (HR) pathway that differentiates itself from all other HR pathways by involving extensive DNA synthesis of up to hundreds of kilobases. This DNA synthesis occurs in G2/M arrested cells by a mechanism distinct from regular DNA replication. BIR initiates by strand invasion of a single end of a DNA double-strand break (DSB) followed by extensive D-loop migration. The main replicative helicase Mcm2-7 is dispensable for BIR, however, Pif1 helicase and its PCNA interaction domain are required. Pif1 helicase was shown to be important for extensive repair-specific DNA synthesis at DSB in budding and fission yeasts, flies, and human cells, implicating conservation of the mechanism. Additionally, Mph1 helicase negatively regulates BIR by unwinding migrating D-loops, and Srs2 promotes BIR by eliminating the toxic joint molecules. Here, we describe the methods that address the following questions in studying BIR: (i) how to distinguish enzymes needed specifically for BIR from enzymes needed for other HR mechanisms that require short patch DNA synthesis, (ii) what are the phenotypes expected for mutants deficient in extensive synthesis during BIR, (iii) how to follow extensive DNA synthesis during BIR? Methods are described using yeast model organism and wild-type cells are compared side-by-side with Pif1 deficient cells.
Details
- Title: Subtitle
- Measuring the contributions of helicases to break-induced replication
- Creators
- Zhenxin Yan - Baylor College of MedicineLiping Liu - University of IowaNhung Pham - Baylor College of MedicinePilendra K Thakre - Baylor College of MedicineAnna Malkova - University of IowaGrzegorz Ira - Baylor College of Medicine
- Resource Type
- Journal article
- Publication Details
- Methods in enzymology, Vol.672, pp.339-368
- DOI
- 10.1016/bs.mie.2022.02.025
- eISSN
- 1557-7988
- Grant note
- DOI: 10.13039/100004917, name: Cancer Prevention and Research Institute of Texas, award: RP210028; DOI: 10.13039/100000002, name: National Institutes of Health, award: GM080600, GM125650, R35GM127006
- Language
- English
- Date published
- 01/01/2022
- Academic Unit
- Biology
- Record Identifier
- 9984296998902771
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