Methods of replicating a DNA molecule for repair of DNA lesion damage or for mutagenesis
AssigneeYeda Research and Development Co. Ltd.
InventorsZvi Livneh, Nina Bacher Reuven, Guy Tomer
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Abstract
UmuC is found to be a translesion replication DNA polymerase which replicates in the presence of UmuD′, RecA and SSB through a DNA lesion of damaged DNA molecule and which is found to be highly mutagenic during in vitro gap-filling replication. A method for replicating a DNA molecule with DNA lesion damage and a method for mutagenesis of a DNA molecule are provided.
Claims38
A method for replicating a DNA molecule having DNA lesion damage, comprising:
- providing a sample containing a DNA molecule with one or more sites of DNA lesion damage;
- and
- contacting the DNA molecule with a translesion replication DNA polymerase, capable of replicating through DNA lesions and selected from the group consisting of UmuC (DNA polymerase V), a fusion protein of UmuC, a fragment of UmuC, and a prokaryotic homologue of UmuC, a fragment of the prokaryotic UmuC homologue, and a fusion protein of the prokaryotic UmuC homologue, in the presence of a combination of UmuD′, RecA and SSB proteins, or in the presence of a combination in which at least one of the UmuD′, RecA and SSB proteins is replaced by a functional prokaryotic homologue thereof, nucleoside 5′-triphosphates, and a divalent metal ion to replicate the damaged DNA molecule by replicating through the one or more sites of DNA lesion damage.
A hybrid protein comprising a fusion of maltose binding protein and UmuC protein.
A method for mutagenesis of a DNA molecule, comprising replicating a DNA molecule with a DNA polymerase selected from the group consisting of UmuC, a fusion protein of UmuC, a fragment of UmuC, a prokaryotic homologue of UmuC, a fragment of the prokaryotic UmuC homologue, and a fusion protein of the prokaryotic UmuC homologue, in the presence of a combination of UmuC′, RecA and SSB proteins, or in the presence of a combination in which at least one of the UmuD′, RecA and SSB proteins is replaced by a functional prokaryotic homologue thereof, nucleoside 5′-triphosphates, and a divalent metal ion to mutagenize the DNA molecule.
Dependencies are inferred from the claim text.