Molecular basis of DNA polymerase ζ’s reverse transcriptase activity

DNA polymerase ζ (Polζ) has emerged as a key reverse transcriptase in RNA-templated double-strand break repair (RT-DSBR), beyond its canonical role in translesion DNA synthesis (TLS). We present here the cryo-EM structure of yeast Polζ (including Rev1) in the act of reverse transcription, wherein the polymerase molds the replicative end of the bound RNA:DNA hybrid into B-like conformation and retains most contacts seen with DNA:DNA. Critically, the polymerase adopts the same conformation as with DNA:DNA, indicating its inherent versatility in accommodating both DNA:DNA and RNA:DNA substrates to preserve genome stability. DNA polymerase ζ (Polζ) functions as a reverse transcriptase in RNA templated double-strand break repair. Here the authors present the cryo-EM structure of Polζ engaged in reverse transcription and reveal that the bound RNA:DNA hybrid is moulded into a B-like conformation while retaining most contacts seen with DNA:DNA.