Abstract:We have been studying the functions of PCNA (proliferating-cell nuclear antigen) for the assembly and reassembly of the replisome during replication fork progression. We have identified the functional interactions between PCNA and several proteins involved in DNA replication and repair from Pyrococcus furiosus. We recently reported that the activity of UDG (uracil-DNA glycosylase) in P. furiosus (PfuUDG) is stimulated by PCNA (PfuPCNA) in vitro, and identified an atypical PCNA-binding site, AKTLF, in the PfuUD… Show more
“…Conversely, PfuPCNA was coprecipitated with PfuAPE by an anti-PfuAPE antibody. We previously showed that PfuUDG and PfuPCNA were coprecipitated with each antibody (46). Figure 4.Physical interaction between PfuAPE and PfuPCNA.…”
Apurinic/apyrimidinic (AP) sites are the most frequently found mutagenic lesions in DNA, and they arise mainly from spontaneous base loss or modified base removal by damage-specific DNA glycosylases. AP sites are cleaved by AP endonucleases, and the resultant gaps in the DNA are repaired by DNA polymerase/DNA ligase reactions. We identified the gene product that is responsible for the AP endonuclease activity in the hyperthermophilic euryarchaeon, Pyrococcus furiosus. Furthermore, we detected the physical interaction between P. furiosus AP endonuclease (PfuAPE) and proliferating cell nuclear antigen (PCNA; PfuPCNA) by a pull-down assay and a surface plasmon resonance analysis. Interestingly, the associated 3′–5′ exonuclease activity, but not the AP endonuclease activity, of PfuAPE was stimulated by PfuPCNA. Immunoprecipitation experiments using the P. furiosus cell extracts supported the interaction between PfuAPE and PfuPCNA in the cells. This is the first report describing the physical and functional interactions between an archaeal AP endonuclease and PCNA. We also detected the ternary complex of PfuPCNA, PfuAPE and Pfu uracil-DNA glycosylase. This complex probably functions to enhance the repair of uracil-containing DNA in P. furiosus cells.
“…Conversely, PfuPCNA was coprecipitated with PfuAPE by an anti-PfuAPE antibody. We previously showed that PfuUDG and PfuPCNA were coprecipitated with each antibody (46). Figure 4.Physical interaction between PfuAPE and PfuPCNA.…”
Apurinic/apyrimidinic (AP) sites are the most frequently found mutagenic lesions in DNA, and they arise mainly from spontaneous base loss or modified base removal by damage-specific DNA glycosylases. AP sites are cleaved by AP endonucleases, and the resultant gaps in the DNA are repaired by DNA polymerase/DNA ligase reactions. We identified the gene product that is responsible for the AP endonuclease activity in the hyperthermophilic euryarchaeon, Pyrococcus furiosus. Furthermore, we detected the physical interaction between P. furiosus AP endonuclease (PfuAPE) and proliferating cell nuclear antigen (PCNA; PfuPCNA) by a pull-down assay and a surface plasmon resonance analysis. Interestingly, the associated 3′–5′ exonuclease activity, but not the AP endonuclease activity, of PfuAPE was stimulated by PfuPCNA. Immunoprecipitation experiments using the P. furiosus cell extracts supported the interaction between PfuAPE and PfuPCNA in the cells. This is the first report describing the physical and functional interactions between an archaeal AP endonuclease and PCNA. We also detected the ternary complex of PfuPCNA, PfuAPE and Pfu uracil-DNA glycosylase. This complex probably functions to enhance the repair of uracil-containing DNA in P. furiosus cells.
“…In addition, as complete genome sequences of three Pyrococcus species became available at a relatively early stage, members of the Thermococcales have been utilized to study a wide range of archaeal biology. These include DNA replication and repair (Hopkins & Paull, 2008;Williams et al, 2008;Yoshimochi et al, 2008;Kiyonari et al, 2009;Mayanagi et al, 2009;Nishida et al, 2009), transcription and its regulation (Vierke et al, 2003;Lee et al, 2005Lee et al, , 2007Goede et al, 2006;Kanai et al, 2007;Santangelo et al, 2007;Hirata et al, 2008a), carbon and energy metabolism (Sapra et al, 2003;Verhees et al, 2003;Siebers & Schönheit, 2005), CRISPR systems (Hale et al, 2009), and cellular responses to stress, such as oxidative (Jenney et al, 1999;Clay et al, 2003), osmotic (Neves et al, 2005;Rodrigues et al, 2007), and temperature stress (Laksanalamai & Robb, 2004;Danno et al, 2008;Fujiwara et al, 2008;Kanzaki et al, 2008;Kida et al, 2008). The abundant genome sequences have also promoted a wealth of '-omics' research including transcriptomics Lee et al, 2006;Trauger et al, 2008), proteomics , structural genomics (Hura et al, 2009), and other genome-based high-throughput strategies (Keese et al, 2010).…”
The tree of life is split into three main branches: eukaryotes, bacteria, and archaea. Our knowledge of eukaryotic and bacteria cell biology has been built on a foundation of studies in model organisms, using the complementary approaches of genetics and biochemistry. Archaea have led to some exciting discoveries in the field of biochemistry, but archaeal genetics has been slow to get off the ground, not least because these organisms inhabit some of the more inhospitable places on earth and are therefore believed to be difficult to culture. In fact, many species can be cultivated with relative ease and there has been tremendous progress in the development of genetic tools for both major archaeal phyla, the Euryarchaeota and the Crenarchaeota. There are several model organisms available for methanogens, halophiles, and thermophiles; in the latter group, there are genetic systems for Sulfolobales and Thermococcales. In this review, we present the advantages and disadvantages of working with each archaeal group, give an overview of their different genetic systems, and direct the neophyte archaeologist to the most appropriate model organism.
“…4 Base excision repair is present in every living organism, even in archaea. 5 A third line of defense exists in this particular kingdom, embedded in the DNA polymerase itself and in the progression of the replication fork. Indeed, archaeal family B DNA polymerases possess an N-terminal domain that can recognize and bind deaminated bases 4 nt upstream the primer-template (P/T) junction during DNA replication.…”
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