Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28

Abstract

Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.

DNA Polymerase ζ without the C-Terminus of Catalytic Subunit Rev3 Retains Characteristic Activity, but Alters Mutation Specificity of Ultraviolet Radiation in Yeast

“…We used standard yeast media with modifications and appropriate carbon sources, as described previously [ 50 ]. Agar, yeast nitrogen base, yeast extract, peptone, glucose are from Formedium, Norfolk, UK.…”

Identification of Potential Phytochemicals Against Cyclin-Dependent Kinase 1 and Cyclin-Dependent Kinase 2: A Molecular Docking and Molecular Dynamic Approach

“…The previously reported Three Dimensional (3D) crystal structure of CDK1 (PDB: 6GU7) [18][19][20][21] having 2.75 Å resolution and CDK2 (PDB: 6GUE) [22][23][24] having 1.99 Å resolution was retrieved from the RCSB Protein data bank [7] . The downloaded structures of the protein were prepared for docking as per the previously described protocol [25] .…”

PCNA Loaders and Unloaders—One Ring That Rules Them All

“…Notably, the Pol Epsilon subunits seem to play a structural role in origin "firing" (initiating bidirectional replication). This is backed by the fact that while deletion of Pol Epsilon is lethal, a mutation in its catalytic region is not; moreover, cells carrying a truncated version of Pol Epsilon with only the region attaching it to the CMG are viable [99]. Therefore, while Pol Delta can presumably replace Pol Epsilon during the replication, the role of Pol Epsilon in origin firing is non-replaceable.…”