Like many proteins in vivo, DNA helicases undergo a variety of post-translational modifications (PTMs) that can impact sub-cellular localization, protein stability, biochemical activity, etc. Mass spectrometry analysis has shown that in Saccharomyces cerevisiae, the PIF1 family helicases Pif1 and Rrm3 are modified by phosphorylation, lysine acetylation, and dimethylation. Using a variety of in vivo and in vitro assays, we’re finding that acetylation by the Esa1 acetyltransferase and deacetylation by the Rpd3 complex affect Pif1’s roles during S-phase of the cell cycle, likely in the Okazaki fragment maturation pathway.
Preliminary data suggest that this regulation is also conserved in the cancer-linked human hPIF1 helicase. We have formed a multi-disciplinary team of collaborators to examine PIF1 PTMs in vivo and in vitro, with current experiments focusing on both PIF1 family helicases, the role of PTMs as molecular switches, and expanding these lines of inquiry to PIF1s in other organisms.
Role of nuclear Pif1 in different biological DNA transactions. Pif1 functions in a flap lengthening during Okazaki fragment processing; b modulating fork convergence during replication termination; c aiding G4 structure resolution; d promoting break-induced replication; e unwinding R-loops; and f regulating telomere length. Acetylation of Pif1 mediated by the lysine acetyltransferase NuA4 (Esa1) and lysine deacetylase (Rpd3) may play a regulatory role in how Pif1′s functions are altered in all of these outlined pathways. Triple arrows indicate directionality of Pif1 activity.
Pif1 Activity is Modulated by DNA Sequence and Structure. Nickens DG, Bochman ML. Biochemistry. 2022 Jan 4;61(1):10-20. doi: 10.1021/acs.biochem.1c00614. Epub 2021 Dec 21. PMID: 34932305
Overcoming stochastic variations in culture variables to quantify and compare growth curve data. Sausen CW, Bochman ML. Bioessays. 2021 Aug;43(8):e2100108. doi: 10.1002/bies.202100108. Epub 2021 Jun 14. PMID: 34128245
Dynamic regulation of Pif1 acetylation is crucial to the maintenance of genome stability. Ononye OE, Sausen CW, Bochman ML, Balakrishnan L. Curr Genet. 2021 Feb;67(1):85-92. doi: 10.1007/s00294-020-01116-5. Epub 2020 Oct 20. PMID: 33079209 Free PMC article. Review.
Lysine acetylation regulates the activity of nuclear Pif1. Ononye OE, Sausen CW, Balakrishnan L, Bochman ML. J Biol Chem. 2020 Nov 13;295(46):15482-15497. doi: 10.1074/jbc.RA120.015164. Epub 2020 Sep 2. PMID: 32878983 Free PMC article.
The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain. Nickens DG, Sausen CW, Bochman ML. Genes (Basel). 2019 May 28;10(6):411. doi: 10.3390/genes10060411. PMID: 31142053 Free PMC article.
The Saccharomyces cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro. Nickens DG, Rogers CM, Bochman ML. J Biol Chem. 2018 Sep 14;293(37):14481-14496. doi: 10.1074/jbc.RA118.004092. Epub 2018 Aug 1. PMID: 30068549 Free PMC article.
The WYL Domain of the PIF1 Helicase from the Thermophilic Bacterium Thermotoga elfii is an Accessory Single-Stranded DNA Binding Module. Andis NM, Sausen CW, Alladin A, Bochman ML. Biochemistry. 2018 Feb 20;57(7):1108-1118. doi: 10.1021/acs.biochem.7b01233. Epub 2018 Jan 30. PMID: 29341597