The SOS Error-Prone DNA Polymerase V Mutasome and β-Sliding Clamp Acting in Concert on Undamaged DNA and during Translesion Synthesis

Abstract

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.

Sending out an SOS - the bacterial DNA damage response

“…In accordance, this is the most studied TLS-polymerase, with orthologs identified in diverse prokaryotes and mobile genetic elements ( Vaisman et al, 2012 ), such as the homologs mucAB described in plasmids ( Perry and Walker, 1982 ) and rumAB in integrative and conjugative elements (ICEs) ( Kulaeva et al, 1995 ). The function of UmuDC was first observed in the 70s, by Miroslav Radman and Evelyn Witkin (Radman, 1974 ; Sikand et al, 2021 ), although at that time the specific polymerase responsible for the mutagenic activity in the SOS response had not been elucidated ( Sikand et al , 2021 ). Genetic identification of umuDC genes was first reported in a search for E. coli strains lacking UV-inducible mutagenesis ( Kato and Shinoura, 1977 ).…”

“…Additionally, early genetic studies have shown that RecA performs a third role in umuDC -dependent SOS mutagenesis ( Blanco et al, 1982 ; Nohmi et al, 1988 ; Dutreix et al, 1989 ; Sweasy et al, 1990 ). In vitro experiments have shown that RecA bound to ssDNA is necessary for mutagenesis, with latest models suggesting that the “mutasome” complex operating in TLS is a molecular assembly of UmuD’ 2 C-RecA-ATP (reviewed by Fujii and Fuchs 2020 ; Jaszczur et al, 2016 ; Sikand et al, 2021 ). RecFOR proteins also have a role in the formation of the RecA filament necessary for UmuD’ 2 C TLS ( Fujii et al , 2006 ).…”

Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

“…The pCol1B9 D37712 plasmid carried a locus that was absent from pCol1B9 4/74 , namely the impC-umuCD operon ( Fig. S3 ) which encodes the error-prone DNA polymerase V responsible for the increased mutation rate linked to the SOS stress response in E. coli (Sikand et al 2021 ).…”

Salmonella entericaserovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

“…The pCol1B9 D37712 plasmid carried a locus that was absent from pCol1B9 4/74 , namely the impC-umuCD operon (Fig. S3) which encodes the error-prone DNA polymerase V responsible for the increased mutation rate linked to the SOS stress response in E. coli (Sikand et al, 2021).…”