Читать книгу Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies - Kenneth N Kreuzer - Страница 26

•The bacteriophage T7 replisome consists of only four proteins: the helicase/primase, ssDNA-binding protein, and DNA polymerase with its host thioredoxin partner.

•T7 DNA polymerase has separate active sites for polymerization and proofreading exonuclease activities.

•Proofreading exonuclease greatly reduces the mutation rate by removing a large majority of misinserted nucleotide residues.

•Processivity of T7 DNA polymerase is increased by the thiore-doxin protein partner, interaction with the T7 helicase/primase, and rapid rebinding of polymerase when it dissociates from the 3′ end.

•T7 helicase/primase unwinds DNA by translocating along a single-strand in a “hand-over-hand” manner, whereby each subunit of the hexamer shifts sequentially from the back to the front of the lock-washer-like structure.

•T7 DNA polymerase stimulates the unwinding activity of the helicase/primase complex, and thus the two proteins are mutually reinforcing.

•The helicase/primase complex travels along the lagging-strand template in the T7 system.

•The replisome functions with a looped lagging strand, and both leading- and lagging-strand polymerases are associated with the helicase/primase complex at the fork.

•The T7 replisome functions as a well-coordinated protein machine, with communication between the leading- and lagging-strand polymerases.

•A structural model of the functioning replisome shows the unwinding point of the parental DNA within the leading-strand polymerase, a flexible DNA region that allows looping between the helicase/primase and the lagging-strand polymerase, and a third DNA polymerase that appears to function as a spare to allow polymerase switching on the lagging strand.

•All ssDNA at the replication fork is coated with ssDNA-binding protein, which helps to organize the replisome.

•Discontinuities in the lagging strand are repaired by the action of a specialized T7 nuclease, DNA polymerase, and DNA ligase.

•A transcript synthesized by T7 RNA polymerase is needed to initiate DNA replication, likely via R-loop formation.