Epigenetic DNA Modification N⁶-Methyladenine Inhibits DNA Replication by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Abstract

N 6 -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 − . The 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 the 6mA:T pair rather than the 6mA:C mispair and preferred to extend beyond Hyp:C rather than the 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 (k pol /K d,dNTP ) was decreased for dTTP incorporation opposite 6mA and dCTP incorporation opposite Hyp compared with dTTP incorporation opposite A. 6mA reduced the incorporation rate (k pol ), and Hyp increased the dissociation constant (K d,dNTP ). However, 6mA or Hyp on template did not affect the binding of DNA polymerase to DNA in binary or ternary complexes. This work provides new insight into the inhibited effects of epigenetic modification of 6mA on DNA replication in PaP1.

Aberrant DNA N6‐methyladenine incorporation via adenylate kinase 1 is suppressed by ADAL deaminase‐dependent 2′‐deoxynucleotide pool sanitation

“…Despite its low abundance, eukaryotic DNA 6mdA plays a number of potential roles in the regulation of gene transcription (Fu et al , 2015; Greer et al , 2015), transposon activity (Zhang et al , 2015; Wu et al , 2016; Liang et al , 2018), nucleosome occupancy (Wang et al , 2017a; Beh et al , 2019), and transgenerational progression (Luo & He, 2017; Ma et al , 2019). Biochemically, the presence of the N 6 ‐methyl group on adenine of the DNA template induces RNA pol II pausing (Wang et al , 2017b) and partially inhibits DNA replication (Li et al , 2019).…”

The deaminase ADAL-pivoted catabolism checkpoint suppresses aberrant DNA N6-methyladenine incorporation

“…It is reasonable that, under physiological conditions, both intracellular and extracellular RNA m6A are tightly regulated by the catabolism checkpoint. Biochemically, the N 6 -methyladenine modi cation on the DNA template disturbs base pairing 54,55 , hinders new chain extension, affects replication 24 and transcription 23 , and regulates related gene expression 22,54 . Therefore, a strict catabolism checkpoint of N 6 -methyladenine is required to maintain genome integrity.…”

“…Despite its low abundance, eukaryotic DNA 6mA has a number of potential functions regulating gene transcription 5,8 , transposon activity 9,10,16 , nucleosome occupancy 6, 17,18 , and transgenerational progression [19][20][21][22] . Biochemically, the presence of the N 6 -methyl group on adenine of the DNA template induces RNA pol II pausing 23 and partially inhibits DNA replication 24 .…”