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Replisome bypass of a protein-based R-loop block by Pif1

Journal Article


Abstract


  • Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Publication Date


  • 2020

Citation


  • Schauer, G. D., Spenkelink, L. M., Lewis, J. S., Yurieva, O., Mueller, S. H., van Oijen, A. M., & O¿Donnell, M. E. (2020). Replisome bypass of a protein-based R-loop block by Pif1. Proceedings of the National Academy of Sciences of the United States of America, 117(48), 30354-30361. doi:10.1073/pnas.2020189117

Scopus Eid


  • 2-s2.0-85097210283

Start Page


  • 30354

End Page


  • 30361

Volume


  • 117

Issue


  • 48

Abstract


  • Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Publication Date


  • 2020

Citation


  • Schauer, G. D., Spenkelink, L. M., Lewis, J. S., Yurieva, O., Mueller, S. H., van Oijen, A. M., & O¿Donnell, M. E. (2020). Replisome bypass of a protein-based R-loop block by Pif1. Proceedings of the National Academy of Sciences of the United States of America, 117(48), 30354-30361. doi:10.1073/pnas.2020189117

Scopus Eid


  • 2-s2.0-85097210283

Start Page


  • 30354

End Page


  • 30361

Volume


  • 117

Issue


  • 48