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Role of RECQ5 Helicase in MUS81-mediated Resolution of Aberrant Replication Intermediates


Di Marco, Stefano. Role of RECQ5 Helicase in MUS81-mediated Resolution of Aberrant Replication Intermediates. 2016, University of Zurich, Faculty of Science.

Abstract

RECQ5 is a member of the RecQ DNA helicase family, a group of highly conserved proteins that are essential for the maintenance of genomic stability. It has been shown that RECQ5 interacts with RAD51 recombinase and disrupts RAD51-ssDNA filaments to suppress aberrant homologous recombination events. The aim of this thesis was to explore the functional significance of the physical interaction of RECQ5 with the MUS81-EME1 endonuclease, which is known to process late replication intermediates at common fragile sites (CFSs) during early mitosis to facilitate chromosome segregation. We have found that RECQ5 associates with CFS loci in a manner dependent on MUS81 and is required for the appearance of MUS81- dependent chromatid breaks/gaps at CFSs following replication stress, a phenomenon termed "expression of CFSs", which is required for proper sister chromatid disjunction. Accordingly, RECQ5-depleted cells showed increased frequency of anaphase bridges and micronuclei, and accumulation of CFS-associated 53BP1 nuclear bodies in G1 cells, phenotypes related to defective chromosome segregation. Moreover, we found that mutational inactivation of the helicase or RAD51-binding domains of RECQ5 increased binding of RAD51 to CFSs, impaired their expression and led to aberrant sister chromatid separation. Consistent with these findings, RECQ5 was found to counteract the inhibitory effect of RAD51 on 3’- flap cleavage by MUS81-EME1 in vitro. Lastly, we have found that expression of CFSs is dependent on phosphorylation of RECQ5 by CDK1 at Ser727. These results suggest that RECQ5 disrupts RAD51 filaments formed on stalled replication forks at CFS and hence facilitates their processing by the MUS81-EME1 endonuclease for proper sister chromatid disjunction during mitosis.

Abstract

RECQ5 is a member of the RecQ DNA helicase family, a group of highly conserved proteins that are essential for the maintenance of genomic stability. It has been shown that RECQ5 interacts with RAD51 recombinase and disrupts RAD51-ssDNA filaments to suppress aberrant homologous recombination events. The aim of this thesis was to explore the functional significance of the physical interaction of RECQ5 with the MUS81-EME1 endonuclease, which is known to process late replication intermediates at common fragile sites (CFSs) during early mitosis to facilitate chromosome segregation. We have found that RECQ5 associates with CFS loci in a manner dependent on MUS81 and is required for the appearance of MUS81- dependent chromatid breaks/gaps at CFSs following replication stress, a phenomenon termed "expression of CFSs", which is required for proper sister chromatid disjunction. Accordingly, RECQ5-depleted cells showed increased frequency of anaphase bridges and micronuclei, and accumulation of CFS-associated 53BP1 nuclear bodies in G1 cells, phenotypes related to defective chromosome segregation. Moreover, we found that mutational inactivation of the helicase or RAD51-binding domains of RECQ5 increased binding of RAD51 to CFSs, impaired their expression and led to aberrant sister chromatid separation. Consistent with these findings, RECQ5 was found to counteract the inhibitory effect of RAD51 on 3’- flap cleavage by MUS81-EME1 in vitro. Lastly, we have found that expression of CFSs is dependent on phosphorylation of RECQ5 by CDK1 at Ser727. These results suggest that RECQ5 disrupts RAD51 filaments formed on stalled replication forks at CFS and hence facilitates their processing by the MUS81-EME1 endonuclease for proper sister chromatid disjunction during mitosis.

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Additional indexing

Item Type:Dissertation
Referees:Lopes Massimo, Janscak Pavel, Cejka Petr, Pichierri Pietro
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:06 Sep 2016 12:13
Last Modified:08 Dec 2017 20:19

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