The yeast Dbf4 Zn2+ finger domain suppresses single-stranded DNA at replication forks initiated from a subset of origins

Jeff Bachant,Elizabeth A Hoffman,Wenyi Feng,Chris Caridi,Constance I Nugent

doi:10.1007/s00294-022-01230-6

Abstract

Dbf4 is the cyclin-like subunit for the Dbf4-dependent protein kinase (DDK), required for activating the replicative helicase at DNA replication origin that fire during S phase. Dbf4 also functions as an adaptor, targeting the DDK to different groups of origins and substrates. Here we report a genome-wide analysis of origin firing in a budding yeast mutant, dbf4-zn, lacking the Zn2+ finger domain within the C-terminus of Dbf4. At one group of origins, which we call dromedaries, we observe an unanticipated DNA replication phenotype: accumulation of single-stranded DNA spanning ± 5kbp from the center of the origins. A similar accumulation of single-stranded DNA at origins occurs more globally in pri1-m4 mutants defective for the catalytic subunit of DNA primase and rad53 mutants defective for the S phase checkpoint following DNA replication stress. We propose the Dbf4 Zn2+ finger suppresses single-stranded gaps at replication forks emanating from dromedary origins. Certain origins may impose an elevated requirement for the DDK to fully initiate DNA synthesis following origin activation. Alternatively, dbf4-zn may be defective for stabilizing/restarting replication forks emanating from dromedary origins during replication stress.

Highlights

Genome duplication is achieved through a tightly regulated program of DNA replication origin (ORI) firing (Raghuraman and Brewer 2010; Marchal et al 2019)
Based on the similarity of this phenotype to rad53 mutants and pri1-M4 mutants defective for the catalytic subunit of DNA primase, we suggest dromedary ORIs either impose an elevated requirement for the dependent protein kinase (DDK) to fully initiate DNA replication, or that the Dbf4 Zn2+ finger contributes to functions that maintain coupling between leading and lagging strand synthesis during replication stress
Strains for these datasets were generated by transforming dbf4-∆ or rad53-21 dbf4-∆ cells with low copy plasmids expressing either DBF4, dbf4-zn or dbf4D3 under control of the endogenous promoter, referred to here as wild type (WT), dbf4-zn, rad53-21, rad53-21 dbf4-zn and rad53 dbf-D3 strains. dbf4-zn is an internal deletion of amino acids 660–688 that form the Z n2+ finger within motif C. dbf4-D3 contains a R701G mutation in a 26 amino acid C-terminal extension beyond the Z n2+ finger. dbf4-zn and dbf4-D3 behave as recessive loss of function mutations, with dbf4-zn exhibiting more severe phenotypes than dbf4-D3

Summary

Introduction

Genome duplication is achieved through a tightly regulated program of DNA replication origin (ORI) firing (Raghuraman and Brewer 2010; Marchal et al 2019). ORIs in some nuclear regions fire early in S phase, while others fire late, leading to a defined spatiotemporal sequence for replicating chromosomes. An essential protein involved in controlling ORI firing is Dbf, the activating subunit for Cdc, the budding yeast Dbf4-dependent protein kinase (DDK; Jackson et al 1993). Ubiquitin-mediated proteolysis in a cyclin-like manner, with Dbf accumulating in G1, peaking during the early S phase and declining at the metaphase to anaphase transition (Cheng et al 1999; Weinreich and Stillman 1999; Godinho Ferreira et al 2000). Activation of each ORI that fires during S phase requires cis-acting DDK phosphorylation of ORI-bound Mcm hexamers (Labib 2010). It has emerged that competition between ORIs for a limiting pool of active DDKis a key determinant of when and how efficiently different ORIs fire (Boos and Ferreira 2019)

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