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The roles of hMSH4-hMSH5 and hMLH1-hMLH3 in meiotic double strand break repair

Soukup, Randal J
http://rave.ohiolink.edu/etdc/view?acc_num=osu1480641841905001

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
The DNA double strand break is a highly cytotoxic DNA lesion. Mouse and human mitotically dividing cells experience ~10 double strand breaks (DSBs) per day that are often repaired through non-homologous end joining and result in the accumulation of short deletions. However, in prophase I of meiosis, ~400 double strand breaks are introduced into primary mouse spermatocytes by the endonuclease SPO11. The cell undergoes a cell-wide DSB repair response which functions to repair each break, and in doing so, pair homologous chromosomes for segregation at the outset of meiosis I. This process generates genetic crossovers between the homologous chromosomes, which are required for accurate chromosome segregation and are also the basis for the reshuffling of genes between maternal and paternal chromosomes. At the center of this DNA repair process is the Holliday Junction, which physically links homologous chromosomes and whose resolution defines the outcome to a genetic crossover or gene conversion event. A number of proteins involved in mitotic DSB repair are also involved with the meiotic process. However, MSH4-MSH5 and MLH1-MLH3 proteins appear to have unique roles in establishing homologous chromosome pairing and segregation during meiotic DSB repair, but do not play any role in mitotic DSB repair. Here we used purified hMSH4-hMSH5 to conduct a series of binding experiments with numerous Holliday Junction constructs. We demonstrate binding of mobile, as well as immobile, Holliday Junctions by hMSH4-hMSH5, and the ability to retain ATP bound hydrolysis-independent sliding clamps on a blocked-end mobile Holliday Junction. In addition, we show that the binding of hMSH4-hMSH5 does not appear to distinguish between the stacked-X or planar conformations of the Holliday Junction. The rate of bulk branch migration by an assembled Holliday Junction did not appear to be affected by the addition of hMSH4-hMSH5. The development of a single molecule approach is reported and will ultimately be used to determine whether the protein transiently or kinetically influences branch migration of individual Holliday Junctions. With no protein currently identified that functions to maintain homologous chromosome pairing through segregation or perform the required Holliday Junction resolution prior to segregation, we partially purified and examined the hMLH1-hMLH3 heterodimer that has been shown to be associated with the development of homologous chromosome linkages. Our preparation of hMLH1-hMLH3 does not appear to display any endonuclease activity or stable complex formation with hMSH4-hMSH5. As has been previously reported we do observe an aggregate that appears to form between hMLH1-hMLH3 and Holliday Junctions at very low ionic strengths. Further hMLH1-hMLH3 purification is required for more complex studies to be performed.
Richard Fishel, PhD (Advisor)
Charles Bell, PhD (Committee Member)
Mark Parthun, PhD (Committee Member)
Mamuka Kvaratskhelia, PhD (Committee Member)
106 p.
Biochemistry; Biophysics; Molecular Biology
hMSH4-hMSH5; Double Strand Break Repair; Holliday Junction; Branch Migration; Biophysics; Molecular Biology

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Citations

  • Soukup, R. J. (2016). The roles of hMSH4-hMSH5 and hMLH1-hMLH3 in meiotic double strand break repair [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480641841905001

    APA Style (7th edition)

  • Soukup, Randal. The roles of hMSH4-hMSH5 and hMLH1-hMLH3 in meiotic double strand break repair. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1480641841905001.

    MLA Style (8th edition)

  • Soukup, Randal. "The roles of hMSH4-hMSH5 and hMLH1-hMLH3 in meiotic double strand break repair." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480641841905001

    Chicago Manual of Style (17th edition)

osu1480641841905001
337
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This open access ETD is published by The Ohio State University and OhioLINK.