Mapping ubiquitination sites of S. cerevisiae Mcm10

Biochemistry and Biophysics Reports
Tianji ZhangAnja-Katrin Bielinsky

Abstract

Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.

Citations

Feb 22, 2017·Genes·Ryan M Baxley, Anja-Katrin Bielinsky

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Methods Mentioned

BETA
co-immunoprecipitation
ubiquitination
two-hybrid
di-ubiquitination
affinity purification
pull-down

Software Mentioned

PEAKS
PEAKS® Studio
Scaffold
Proteome
PEAKS®
Sequest

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