Ishino, S. | Search bioRxiv

bioRxiv2024

Chromosomal domain formation by archaeal SMC, a roadblock protein, and DNA structure

Structural maintenance of chromosomes (SMC) complexes fold genomes by extruding DNA loops. In eukaryotes, loop-extruding SMC complexes form topologically associating domains (TADs) by being stalled by roadblock proteins. It remains unclear whether a similar mechanism of domain formation exists in prokaryotes. Using high-resolution chromosome conformation capture sequencing, we show that an archaeal homolog of the bacterial Smc-ScpAB complex organizes the genome of Thermococcus kodakarensis into TAD-like domains. We also find that TrmBL2, a nucleoid- associated protein that forms a stiff nucleoprotein filament, stalls the T. kodakarensis SMC complex and establishes a boundary at the site-specific recombination site dif. TrmBL2 stalls the SMC complex at tens of additional non-boundary loci with lower efficiency. Intriguingly, the stalling efficiency is correlated with structural properties of underlying DNA sequences. Our study illuminates not only a eukaryotic-like mechanism of domain formation in archaea, but also an unforeseen role of intrinsic DNA structure in large-scale genome organization.

molecular biology↗

bioRxiv2021

A well conserved archaeal B-family polymerase functions as a mismatch and lesion extender

B-family DNA polymerases (PolBs) of different groups are widespread in Archaea and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that are poorly characterized is PolB2 whose members occur in many archaea but are predicted as an inactivated form of DNA polymerase. Herein, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase harbors a robust nucleotide incorporation activity, but devoid of the 3-5 exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. Together, these data suggested Dpo2 functions as a mismatch and lesion extender, representing a novel type of PolB that is primarily involved in DNA damage repair in Archaea. Insights were also gained into the functional adaptation of the motif C in the mismatch extension of the B-family DNA polymerases.

microbiology↗