International Journal of Molecular Sciences

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Molecular Mechanisms of Archaea

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Dear Colleagues,

Genomic integrity is a fundamental requirement for the proper functioning of all cellular processes and for the transmission of genetic information to offspring. It is true that random genome mutations are the driving force of biodiversity and guide the evolution of living organisms; however genomic instability proves detrimental in most cases.

DNA damage can be caused by exogenous sources (i.e. chemical cross-linkers or UV-light), but also by endogenous errors during replication, recombination, and chromosome segregation as the incorporation of mismatch errors, torsional stress of the DNA helix, and spontaneous deamination or hydrolysis. To counteract damage all organisms have developed several DNA repair pathways and the choice among them is linked to the type of DNA damage and/or cell cycle phases. Compared to Eukarya and Bacteria, Archaea must have more robust DNA replication and repair complexes to ensure genome fidelity due to the environmental stresses they usually live under. Since archaeal enzyme structures provide critical data about the architecture and mechanisms of key DNA repair proteins, the study of archaeal structural biology is fundamental for both medical and industrial impacts. With this Special Issue of IJMS we aim to enlarge current knowledge on molecular mechanisms of DNA replication and recombinational repair, particularly in Archaea which have been considered the most efficient model of DNA repair pathways for decades now. We hope to contribute to the knowledge in DNA repair mechanisms and consequently to identify new strategies for the most accurate treatments in human diseases.

Dr. Mariarosaria De Falco
Dr. Mariarita De Felice
Guest Editors

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Keywords

DNA replication
DNA repair
DNA damage response (DDR)
genome stability
archaea
homologous recombination

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Research

17 pages, 10483 KiB

Open AccessArticle

Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon

by Johanna Thomsen and Ruth A. Schmitz

Int. J. Mol. Sci. 2022, 23(19), 11910; https://doi.org/10.3390/ijms231911910 - 07 Oct 2022

Viewed by 2460

Abstract

Due to their role in methane production, methanoarchaea are of high ecological relevance and genetic systems have been ever more established in the last two decades. The system for protein expression in Methanosarcina using a comprehensive shuttle vector is established; however, details about its replication mechanism in methanoarchaea remain unknown. Here, we report on a significant optimisation of the rather large shuttle vector pWM321 (8.9 kbp) generated by Metcalf through a decrease in its size by about 35% by means of the deletion of several non-coding regions and the ssrA gene. The resulting plasmid (pRS1595) still stably replicates in M. mazei and—most likely due to its reduced size—shows a significantly higher transformation efficiency compared to pWM321. In addition, we investigate the essential gene repA, coding for a rep type protein. RepA was heterologously expressed in Escherichia coli, purified and characterised, demonstrating the significant binding and nicking activity of supercoiled plasmid DNA. Based on our findings we propose that the optimised shuttle vector replicates via a rolling circle mechanism with RepA as the initial replication protein in Methanosarcina. On the basis of bioinformatic comparisons, we propose the presence and location of a double-strand and a single-strand origin, which need to be further verified. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Archaea)

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14 pages, 6951 KiB

Open AccessArticle

Mechanism Underlying the Bypass of Apurinic/Pyrimidinic Site Analogs by Sulfolobus acidocaldarius DNA Polymerase IV

by Qin-Ying Huang, Dong Song, Wei-Wei Wang, Li Peng, Hai-Feng Chen, Xiang Xiao and Xi-Peng Liu

Int. J. Mol. Sci. 2022, 23(5), 2729; https://doi.org/10.3390/ijms23052729 - 01 Mar 2022

Viewed by 1582

Abstract

The spontaneous depurination of genomic DNA occurs frequently and generates apurinic/pyrimidinic (AP) site damage that is mutagenic or lethal to cells. Error-prone DNA polymerases are specifically responsible for the translesion synthesis (TLS) of specific DNA damage, such as AP site damage, generally with relatively low fidelity. The Y-family DNA polymerases are the main error-prone DNA polymerases, and they employ three mechanisms to perform TLS, including template-skipping, dNTP-stabilized misalignment, and misincorporation-misalignment. The bypass mechanism of the dinB homolog (Dbh), an archaeal Y-family DNA polymerase from Sulfolobus acidocaldarius, is unclear and needs to be confirmed. In this study, we show that the Dbh primarily uses template skipping accompanied by dNTP-stabilized misalignment to bypass AP site analogs, and the incorporation of the first nucleotide across the AP site is the most difficult. Furthermore, based on the reported crystal structures, we confirmed that three conserved residues (Y249, R333, and I295) in the little finger (LF) domain and residue K78 in the palm subdomain of the catalytic core domain are very important for TLS. These results deepen our understanding of how archaeal Y-family DNA polymerases deal with intracellular AP site damage and provide a biochemical basis for elucidating the intracellular function of these polymerases. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Archaea)

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