Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae

Abstract

B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here, we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

The potential role of COVID-19 in the induction of DNA damage

“…The defects in the replication fork can promote tumorigenesis by inducing genetic instability [ 41 ]. DNA polymerase δ is one of the most important enzymes for genome stability; it is the main polymerase involved in the synthesis of the lagging strand during replicative DNA synthesis and is one of several enzymes involved in DNA repair mechanisms [ [42] , [43] , [44] ]. Thus, the effects of nsp13 on DNA polymerase δ could contribute not only to direct DNA damage due to replication fork stress, but can potentially also promote genome instability in the presence of other environmental factors, such as air pollution that is associated with the severity of COVID-19 disease and with DNA damage [ 45 , 46 ].…”

Revised Mechanism of Hydroxyurea Induced Cell Cycle Arrest and an Improved Alternative

“…Pol α contains conserved CysA and CysB motifs in the CTD of its polymerase domain, but it remains unclear whether they coordinate Fe-S clusters (46, 55). This uncertainty is in large part due to the difficulty of purifying these complexes while retaining labile cofactors, however, genetics studies like those performed for Pol δ (59, 60) and Pol ε (56) can be informative regarding the importance of these metal binding sites. Pol α is essential, making it highly sensitive to changes in activity.…”

“…Further, disruption of the Fe-S coordination by CysB mutation in the CTD of Pol3 (the polymerase subunit of Pol δ, the 3-subunit lagging strand Pol) destabilized the complex and similarly compromised both its polymerase and exonuclease activities (55,58). The effect on Pol δ was also observed in vivo by Vernis and colleagues, where mutation of its CTD CysB domain resulted in genome instability (59), corroborating earlier results from a genetic screen (60). This group has also demonstrated that HU activates the oxidative stress response pathway, which attenuates the deleterious oxidative effects of HU in part by upregulation of two critical genes involved in Fe-S biosynthesis, and additionally that H2O2 inhibits Leu1, an isopropylmalate isomerase containing a Fe-S cluster (61); these findings collectively led to speculation that various stress and/or anti-cancer drugs could be responsible for similar effects by oxidizing Fe-S clusters in cells (59,61).…”

“…Still, we note that in theory, inhibition of any of the replicative polymerases would be enough to stall the cell cycle. The fact that perturbation of CysB in Pol δ and CysX in Pol ε inhibits these critical polymerases in vivo (57, 59, 60), and similarly that primase activity relies on Fe-S coordination (48, 51) indicates that oxidization of the integral Fe-S cluster in these complexes would be sufficient to stall cells. Critically, it was shown that mutation of the Cys residues that coordinate Fe-S in the CTD of human Pri2 (the regulatory subunit of primase) results in defective Pol α loading and initiation, with a temperature sensitive phenotype of G1/S arrest (80).…”

COVID-19: An overview on possible transmission ways, sampling matrices and diagnosis

“…These events prompt a high uptake of metabolites necessary for viral replication. [7][8][9] It is noteworthy that the mere expression of nsp13 is adequate to cause fork stress and DNA damage, even without any other viral constituents or viral replication. 7 Coronaviruses elicit cell cycle arrest in the S-phase by inducing DNA replication fork stress, thereby increasing the uptake of vital metabolites necessary for viral replication.…”