High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase

Jaime Iranzo,Susana M Cerritelli,Andrei Chabes,Aziz El Hage,David Tollervey,Robert J Crouch,Sushma Sharma

doi:10.1093/nar/gkaa103

Abstract

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

Highlights

In eukaryotes, undamaged nuclear DNA is replicated by three members of the B family of DNA polymerases (Pols), Pol ␣, Pol ε and Pol ␦, whose catalytic subunits are Pol1, Pol2 and Pol3, respectively
If a threshold of single genomic ribonucleoside monophosphates (rNMPs) is exceeded in absence of RER and presence of limited deoxyribonucleoside tri-phosphates (dNTPs) pools, Top1mediated genome instability leads to severe growth defects
As a control of formamide-denaturation of genomic DNA, both the WT strain and the single mutant PGAL:3HA-RNR1 (Rnr1 [−]) showed fragmentation of their genomic DNA after incubation with the DNA nicking endonuclease Nb.BtsI (Supplementary Figure S5, compare samples 1 with 4, and 9 with 12). These results indicate that most Alkali-fragments from total DNA (Afts) detected in PGAL:3HA-RNR1 rnh201Δ (Rnr1 [−]) mutants without or with an ribonucleoside triphosphates (rNTPs)-permissive Pol are due to cleavage at sites of genome-embedded ribonucleotides (Figure 5A, lower-part of Figure 5B, Supplementary Figure S4B, Figure 5C and Supplementary Table S5, samples 4, 8, 10, 14, 16, 20 and 22)

Summary

Introduction

In eukaryotes, undamaged nuclear DNA is replicated by three members of the B family of DNA polymerases (Pols), Pol ␣, Pol ε and Pol ␦, whose catalytic subunits are Pol, Pol and Pol, respectively (for a review, see e.g. [1]). Pol ␣-RNA primase complex initiates synthesis of both leading and lagging strands. Pol ␣ is replaced by Pol ε, which synthesizes long stretches of DNA in a processive manner. Pol ␦ takes over from Pol ␣ and synthesizes Okazaki fragments ( referred to as ‘OF’), which are short segments of about 200 nt that are processed and ligated after polymerization The stringency of selection against the incorporation of ribonucleoside monophosphates (rNMPs) varies among replicative Pols ␣, ε and ␦ ((10); for reviews, see e.g. As cellular rNTP concentrations in eukaryotes are generally one to two orders of magnitude higher than those of the corresponding dNTPs, this potentially affects the frequencies of rNMP incorporation by the replicative Pols [10,13]

Methods

Results

Conclusion