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N-methyladenine (6mA), a newly identified epigenetic modification, plays important roles in regulation of various biological processes. However, the effect of 6mA on DNA replication has been little addressed. In this work, we investigated how 6mA affected DNA replication by DNA polymerase of Pseudomonas aeruginosa Phage PaP1 (gp90 exo-). The presence of 6mA, as well as its intermediate hypoxanthine (Hyp), inhibited DNA replication by gp90 exo-. 6mA reduced dTTP incorporation efficiency by 10-fold and inhibited next-base extension efficiency by 100-fold. Differently, dCTP was preferentially incorporated opposite Hyp among four dNTPs. Gp90 exo- reduced the extension priority beyond 6m
T pair rather than 6m
C mispair and preferred to extend beyond Hyp:C rather than Hyp:T pair. Incorporation of dTTP opposite 6mA and dCTP opposite Hyp showed fast burst phases. The burst rate and burst amplitude were both reduced for 6mA compared with unmodified A. Moreover, the total incorporation efficiency (kpol/Kd,dNTP) was decreased for dTTP incorporation opposite 6mA and dCTP incorporation opposite Hyp compared with dTTP incorporation opposite A. 6mA reduced the incorporation rate (kpol) and Hyp increased the dissociation constant (Kd,dNTP). However, 6mA or Hyp on template did not affect the binding of DNA polymerase to DNA in binary or ternary complex. This work provides new insight in the inhibited effects of epigenetic modification 6mA on DNA replication in PaP1.
This article was published in the following journal.
Name: Chemical research in toxicology
N-methyladenine (6mA), as a newly reported epigenetic marker, plays significant roles in regulation of various biological processes in eukaryotes. However, the effect of 6mA on human DNA replication r...
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Antibiotic pigment produced by Pseudomonas aeruginosa.
A DNA (cytosine-5-)-methyltransferase that contains a central CxxC type zinc finger motif. It binds poly(ADP)-ribose and its expression is regulated by POLY (ADP-RIBOSE) POLYMERASE-1. DNMT1 methylates CpG residues, with a preference for hemimethylated DNA, and associates with DNA replication sites in S PHASE to maintain the methylation pattern in the newly synthesized strand, which is essential for EPIGENETIC PROCESSES. It also associates with CHROMATIN during G2 PHASE and MITOSIS to maintain DNA methylation independently of replication. It is responsible for maintaining methylation patterns established in development; mutations in the DNMT1 gene are associated with HEREDITARY SENSORY NEUROPATHY TYPE 1 class E.
A DNA-directed DNA polymerase that functions in the replication of MITOCHONDRIAL DNA. Mutations in the gene that encodes this enzyme (POLG) are associated with some forms of OPHTHALMOPLEGIA, CHRONIC EXTERNAL PROGRESSIVE.
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