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.
Highlights
Bei San Huan Dong Lu, Beijing, P.R
Ubiquitination regulates the binding between Mcm10 and proliferating cell nuclear antigen (PCNA), which is dependent on the cell cycle and a PCNA interacting peptide (PIP) box that is buried in the central domain of Mcm10 [15]
The evolutionarily conserved replication factor Mcm10 is mono-ubiquitinated at two distinct lysines, and only the ubiquitinated form of the protein binds to PCNA [15]
Summary
Under those conditions, replication forks move slowly and elicit responses triggered by the accumulation of single-stranded (ss) DNA, a byproduct of fork stalling [13]. The PIP box is part of a highly conserved oligonucleotide/-saccharide binding (OB) fold. These β-barrel motifs are common in RNA and DNA binding proteins and form a cleft that allows for the direct interaction with the nucleic acid backbone [16]. Due to the spatial arrangement of the PIP box and the Hsp10-like motifs, the simultaneous interaction of Mcm with pol-α and PCNA seems highly unlikely. We identify lysine (K) 372 as the primary site and map several alternative sites for ubiquitin attachment on Mcm in S. cerevisiae
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