Abstract:The bacterial single-stranded DNA (ssDNA)-binding protein (SSB) and the archaeal/eukaryotic replication protein A (RPA) play multiple and essential roles in almost every aspect of nucleic acid metabolism, including DNA replication, repair, and recombination (14, 25). The SSBs and RPAs across the three domains of life share a common and conserved module called the oligonucleotide/oligosaccharide-binding fold (OB fold) (16). Structurally, the common OB folds consist of fivestranded -sheets coiled to form a clos… Show more
“…borinquense RpaC protein: N- and C-terminal deletions (ΔNTD and ΔCTD respectively), single OB fold deletions (ΔOB A , ΔOB B and ΔOB C ), double OB fold deletions (ΔOB AB , ΔOB BC and ΔOB AC ) and a triple OB deletion (ΔOB ABC ). Note that, in contrast to previously reported biochemical analysis of the M. acetivorans MacRPA1 protein, we chose to delete individual OB fold domains in their entirety rather than create chimeric OB folds by fusing adjacent folds (15). Figure 5.Structure–function analysis of RpaC.…”
Single-stranded DNA-binding proteins (SSBs) play vital roles in all aspects of DNA metabolism in all three domains of life and are characterized by the presence of one or more OB fold ssDNA-binding domains. Here, using the genetically tractable euryarchaeon Haloferax volcanii as a model, we present the first genetic analysis of SSB function in the archaea. We show that genes encoding the OB fold and zinc finger-containing RpaA1 and RpaB1 proteins are individually non-essential for cell viability but share an essential function, whereas the gene encoding the triple OB fold RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Deletion analysis is used to demonstrate important roles for individual OB folds in RpaC and to show that conserved N- and C-terminal domains are required for efficient repair of DNA damage. Consistent with a role for RpaC in DNA repair, elevated expression of this protein leads to enhanced resistance to DNA damage. Taken together, our results offer important insights into archaeal SSB function and establish the haloarchaea as a valuable model for further studies.
“…borinquense RpaC protein: N- and C-terminal deletions (ΔNTD and ΔCTD respectively), single OB fold deletions (ΔOB A , ΔOB B and ΔOB C ), double OB fold deletions (ΔOB AB , ΔOB BC and ΔOB AC ) and a triple OB deletion (ΔOB ABC ). Note that, in contrast to previously reported biochemical analysis of the M. acetivorans MacRPA1 protein, we chose to delete individual OB fold domains in their entirety rather than create chimeric OB folds by fusing adjacent folds (15). Figure 5.Structure–function analysis of RpaC.…”
Single-stranded DNA-binding proteins (SSBs) play vital roles in all aspects of DNA metabolism in all three domains of life and are characterized by the presence of one or more OB fold ssDNA-binding domains. Here, using the genetically tractable euryarchaeon Haloferax volcanii as a model, we present the first genetic analysis of SSB function in the archaea. We show that genes encoding the OB fold and zinc finger-containing RpaA1 and RpaB1 proteins are individually non-essential for cell viability but share an essential function, whereas the gene encoding the triple OB fold RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Deletion analysis is used to demonstrate important roles for individual OB folds in RpaC and to show that conserved N- and C-terminal domains are required for efficient repair of DNA damage. Consistent with a role for RpaC in DNA repair, elevated expression of this protein leads to enhanced resistance to DNA damage. Taken together, our results offer important insights into archaeal SSB function and establish the haloarchaea as a valuable model for further studies.
“…SSBs [also known as RPA (replication protein A) in eukaryotes] act as DNA-damage sensors and ssDNA chaperones in many DNA repair, replication and recombination pathways in all forms of life. SSBs come in many different varieties: homodimers and homotetramers in bacteria, homodimers and heterotrimers in eukarya, and monomers, dimers and trimers in archaea [9]. The core functional domain of the canonical SSB is the OB-fold (oligonucleotide-binding fold), which is a twisted β-barrel with a binding site that accommodates four nucleotides of ssDNA [10].…”
The process of information exchange between two homologous DNA duplexes is known as homologous recombination (HR) or double-strand break repair (DSBR), depending on the context. HR is the fundamental process underlying the genome shuffling that expands genetic diversity (for example during meiosis in eukaryotes). DSBR is an essential repair pathway in all three domains of life, and plays a major role in the rescue of stalled or collapsed replication forks, a phenomenon known as recombination-dependent replication (RDR). The process of HR in the archaea is gradually being elucidated, initially from structural and biochemical studies, but increasingly using new genetic systems. The present review focuses on our current understanding of the structures, functions and interactions of archaeal HR proteins, with an emphasis on recent advances. There are still many unknown aspects of archaeal HR, most notably the mechanism of branch migration of Holliday junctions, which is also an open question in eukarya.
“…M. acetivorans RPA1, 2, and 3 have four, two, and two OBfolds, respectively. The hypothesis that homologous recombination might play an important role in generating the diversity of OB-folds in archaea was proposed, based on experiments characterizing the engineered RPAs with various OB-folds [92].…”
Section: Single-stranded Dna Binding Proteinmentioning
Archaea, the third domain of life, are interesting organisms to study from the aspects of molecular and evolutionary biology. Archaeal cells have a unicellular ultrastructure without a nucleus, resembling bacterial cells, but the proteins involved in genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of Eukaryota. Therefore, archaea provide useful model systems to understand the more complex mechanisms of genetic information processing in eukaryotic cells. Moreover, the hyperthermophilic archaea provide very stable proteins, which are especially useful for the isolation of replisomal multicomplexes, to analyze their structures and functions. This review focuses on the history, current status, and future directions of archaeal DNA replication studies.
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