Replication Repair

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Summary of Work: DNA damage produces cell death or mutation, both of which can have serious health consequences. Most DNA damage is repaired before it can cause harm. The classically defined mechanisms of DNA repair are direct reversal of the damage, excision of the damage followed by resynthesis of the damaged strand, recombinational processes that bypass the damage without removing it, and mutagenic translesion synthesis. A fourth but poorly characterized mechanism was identified in bacteriophage T4. Mutations in certain genes of DNA replication reduce survival after treatments inducing a variety of kinds of DNA damage, but the mutations hardly affect phage reproduction. The process does not involve any of the classical mechanisms and was named "replication repair" because it involves enzymes of DNA replication. We have set up the Alberts-Nossal bacteriophage T4 eight-protein system for replicating both strands of DNA in vitro and have investigated the processing of damaged DNA comparing wild-type and mutant enzymes. A mutant DNA-binding protein exhibits reduced affinity for single-stranded DNA. Using an artificial replication fork, we can observe template switching in which a blocked leading strand can switch to the other daughter strand in order to continue synthesis beyond the block. In this assay, the mutant proteins are strongly defective in promoting strand switching.