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Elucidating the Role of MMS22L-TONSL Heterodimer in Homologous Recombination


Mlejnkova, Lucie Jarmila. Elucidating the Role of MMS22L-TONSL Heterodimer in Homologous Recombination. 2016, University of Zurich, Faculty of Science.

Abstract

Homologous recombination (HR) is a mostly accurate DNA repair pathway, which takes place in the S-phase of the cell cycle. It repairs DNA double-strand breaks (DSBs), DNA single-strand breaks and contributes to the repair of interstrand DNA crosslinks. While the mechanism of a two-ended DSB is relatively well understood, how recombination restores DNA integrity upon replisome stalling or collapse, remains poorly understood.
In yeast, Mms22 was found to promote homologous recombination specifically upon DNA replication fork stalling. In human cells, the human homologue MMS22L (Mms22-like) forms a complex with TONSL (Tonsoku-like). MMS22L-TONSL complex localizes at sites of damaged DNA upon laser microirradiation. Depletion of MMS22L or TONSL caused hypersensitivity to a variety of DNA-damaging drugs that interfere with DNA replication. Furthermore, depletion of MMS22L or TONSL resulted in persistent RPA foci and decreased formation of RAD51 foci. These data suggested that MMS22L-TONSL complex might play a role in the initiation of HR- dependent repair specifically downstream of DNA end resection prior to RAD51 loading.
The aim of this project was to perform biochemical analyses to investigate the potential role of MMS22L-TONSL in HR, in particular as a recombination mediator. Recombination mediators, such as BRCA2 or RAD51 paralogs, help to load RAD51 on RPA-coated ssDNA. RAD51 then performs search for homologous DNA duplex, which subsequently leads to HR repair.
First, we purified MMS22L-TONSL complex from Sf9 insect cell expression system. The complex purified as heterodimer. We then tested its ability to bind various DNA substrates. We found that MMS22L-TONSL complex binds long stretches of DNA, suggesting that DNA might be bound cooperatively. Furthermore, MMS22L-TONSL complex bound RPA-coated ssDNA. Next we tested direct protein- protein interactions between MMS22L-TONSL or MMS22L and RAD51. Strikingly, RAD51 binds both MMS22L-TONSL and MMS22L, suggesting that MMS22L is the interacting subunit. MMS22L-TONSL also facilitates RAD51-dependent DNA strand exchange activity in an ATP-dependent manner by lowering RAD51's binding affinity to dsDNA. As a result, this facilitates RAD51 filament formation on single-stranded DNA when dsDNA is present. Our data collectively support a notion that MMS22L-TONSL acts in homologous recombination as a recombination mediator, similarly as BRCA2 and RAD51 paralogs such as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3.

Abstract

Homologous recombination (HR) is a mostly accurate DNA repair pathway, which takes place in the S-phase of the cell cycle. It repairs DNA double-strand breaks (DSBs), DNA single-strand breaks and contributes to the repair of interstrand DNA crosslinks. While the mechanism of a two-ended DSB is relatively well understood, how recombination restores DNA integrity upon replisome stalling or collapse, remains poorly understood.
In yeast, Mms22 was found to promote homologous recombination specifically upon DNA replication fork stalling. In human cells, the human homologue MMS22L (Mms22-like) forms a complex with TONSL (Tonsoku-like). MMS22L-TONSL complex localizes at sites of damaged DNA upon laser microirradiation. Depletion of MMS22L or TONSL caused hypersensitivity to a variety of DNA-damaging drugs that interfere with DNA replication. Furthermore, depletion of MMS22L or TONSL resulted in persistent RPA foci and decreased formation of RAD51 foci. These data suggested that MMS22L-TONSL complex might play a role in the initiation of HR- dependent repair specifically downstream of DNA end resection prior to RAD51 loading.
The aim of this project was to perform biochemical analyses to investigate the potential role of MMS22L-TONSL in HR, in particular as a recombination mediator. Recombination mediators, such as BRCA2 or RAD51 paralogs, help to load RAD51 on RPA-coated ssDNA. RAD51 then performs search for homologous DNA duplex, which subsequently leads to HR repair.
First, we purified MMS22L-TONSL complex from Sf9 insect cell expression system. The complex purified as heterodimer. We then tested its ability to bind various DNA substrates. We found that MMS22L-TONSL complex binds long stretches of DNA, suggesting that DNA might be bound cooperatively. Furthermore, MMS22L-TONSL complex bound RPA-coated ssDNA. Next we tested direct protein- protein interactions between MMS22L-TONSL or MMS22L and RAD51. Strikingly, RAD51 binds both MMS22L-TONSL and MMS22L, suggesting that MMS22L is the interacting subunit. MMS22L-TONSL also facilitates RAD51-dependent DNA strand exchange activity in an ATP-dependent manner by lowering RAD51's binding affinity to dsDNA. As a result, this facilitates RAD51 filament formation on single-stranded DNA when dsDNA is present. Our data collectively support a notion that MMS22L-TONSL acts in homologous recombination as a recombination mediator, similarly as BRCA2 and RAD51 paralogs such as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3.

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Additional indexing

Item Type:Dissertation
Referees:Cejka Petr, Lopes Massimo, Peter Matthias
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2016
Deposited On:16 Dec 2016 09:59
Last Modified:28 Apr 2017 05:52

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