A DinB variant reveals diverse physiological consequences of incomplete TLS extension by a Y-family DNA polymerase.
The only Y-family DNA polymerase conserved among all domains of life, DinB and its mammalian ortholog pol kappa, catalyzes proficient bypass of damaged DNA in translesion synthesis (TLS). Y-family DNA polymerases, including DinB, have been implicated in diverse biological phenomena ranging from adaptive mutagenesis in bacteria to several human cancers. Complete TLS requires dNTP insertion opposite a replication blocking lesion and subsequent extension with several dNTP additions. Here we report remarkably proficient TLS extension by DinB from Escherichia coli. We also describe a TLS DNA polymerase variant generated by mutation of an evolutionarily conserved tyrosine (Y79). This mutant DinB protein is capable of catalyzing dNTP insertion opposite a replication-blocking lesion, but cannot complete TLS, stalling three nucleotides after an N(2)-dG adduct. Strikingly, expression of this variant transforms a bacteriostatic DNA damaging agent into a bactericidal drug, resulting in profound toxicity even in a dinB(+) background. We find that this phenomenon is not exclusively due to a futile cycle of abortive TLS followed by exonucleolytic reversal. Rather, gene products with roles in cell death and metal homeostasis modulate the toxicity of DinB(Y79L) expression. Together, these results indicate that DinB is specialized to perform remarkably proficient insertion and extension on damaged DNA, and also expose unexpected connections between TLS and cell fate.
Jarosz
Daniel F
DF
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Cohen
Susan E
SE
Delaney
James C
JC
Essigmann
John M
JM
Walker
Graham C
GC
D000900:Anti-Bacterial Agents; D016923:Cell Death / Q000235:genetics; D017124:Conserved Sequence; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology* / Q000633:toxicity; D020125:Mutation, Missense
D000900:Anti-Bacterial Agents; C094399:DinB protein, E coli; D029968:Escherichia coli Proteins; D004259:DNA-Directed DNA Polymerase