Abstract:Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity… Show more
“…After that, the recombinase performs a homology search to find a dsDNA having homologous sequence. The global mechanism is reviewed in [12,56,58]. Briefly, the homology recognition and short strand exchange require binding of ATP, allowing filaments to function as rotary motors.…”
Section: Rada Catalyzes D-loop Formation On a Circular Dna Substratementioning
Among the three domains of life, the process of homologous recombination (HR) plays a central role in the repair of double-strand DNA breaks and the restart of stalled replication forks. Curiously, main protein actors involved in the HR process appear to be essential for hyperthermophilic Archaea raising interesting questions about the role of HR in replication and repair strategies of those Archaea living in extreme conditions. One key actor of this process is the recombinase RadA, which allows the homologous strand search and provides a DNA substrate required for following DNA synthesis and restoring genetic information. DNA polymerase operation after the strand exchange step is unclear in Archaea. Working with Pyrococcus abyssi proteins, here we show that both DNA polymerases, family-B polymerase (PolB) and family-D polymerase (PolD), can take charge of processing the RadA-mediated recombination intermediates. Our results also indicate that PolD is far less efficient, as compared with PolB, to extend the invaded DNA at the displacement-loop (D-loop) substrate. These observations coincide with previous genetic analyses obtained on Thermococcus species showing that PolB is mainly involved in DNA repair without being essential probably because PolD could take over combined with additional partners.
“…After that, the recombinase performs a homology search to find a dsDNA having homologous sequence. The global mechanism is reviewed in [12,56,58]. Briefly, the homology recognition and short strand exchange require binding of ATP, allowing filaments to function as rotary motors.…”
Section: Rada Catalyzes D-loop Formation On a Circular Dna Substratementioning
Among the three domains of life, the process of homologous recombination (HR) plays a central role in the repair of double-strand DNA breaks and the restart of stalled replication forks. Curiously, main protein actors involved in the HR process appear to be essential for hyperthermophilic Archaea raising interesting questions about the role of HR in replication and repair strategies of those Archaea living in extreme conditions. One key actor of this process is the recombinase RadA, which allows the homologous strand search and provides a DNA substrate required for following DNA synthesis and restoring genetic information. DNA polymerase operation after the strand exchange step is unclear in Archaea. Working with Pyrococcus abyssi proteins, here we show that both DNA polymerases, family-B polymerase (PolB) and family-D polymerase (PolD), can take charge of processing the RadA-mediated recombination intermediates. Our results also indicate that PolD is far less efficient, as compared with PolB, to extend the invaded DNA at the displacement-loop (D-loop) substrate. These observations coincide with previous genetic analyses obtained on Thermococcus species showing that PolB is mainly involved in DNA repair without being essential probably because PolD could take over combined with additional partners.
“…On phylogenetic analysis, Gp65 was found to share homology with AAA ATPase protein of cluster F1 mycobacteriophage Gumbie, Saal, and Wachhund (Figure S2). Conserved domain analysis predicted AAA_24 domain (Gp65 13-192 , E value: 9.93e-30) and RecA/RadA domain (Gp65 17-113 , E value: 3.83e-06) involved in replication, recombination or repair of DNA (Figure 1) (del Val et al ., 2019). The predicted domains suggested a possible ATP hydrolysis mediated DNA repair/maintenance role of this protein in SimranZ1 phage.…”
Mycobacteriophages are viruses of Mycobacterium spp. with promising diagnostic and therapeutic potential. Phage genome exploration and characterization of their proteomes are essential to gain a better understanding of their role in phage biology. So far, about 2014 mycobacteriophages have been genomically defined and 1563 phage protein families (phamilies) are identified. However, the function of only a fraction (about 15%) is known and a majority of ORFs in phage genomes are hypothetical proteins. In this study, from the annotated genome of a F1 cluster mycobacteriophage SimranZ1, a putative AAA ATPase (Gp65, Pham 9410) is characterized as a DNA-dependent ATPase. Sequence-based functional annotation predicted Gp65 to belong to the P-loop NTPase superfamily, having AAA_24 and RecA/RadA domains which are known to be involved in ATP-dependent DNA repair/maintenance mechanism. On molecular docking, Gly21 and Ser23 of Gp65 showed specific binding with ATP. Using a microtiter plate assay, ATPase activity of Gp65 was experimentally verified which was found to increase in the presence of dsDNA. Gel electrophoresis under non-denaturing condition showed the oligomeric states of Gp65 and Transmission Electron Microscopy revealed it to exist as a hexamer having a prominent central pore with a diameter of 1.9 nm. In summary, functional characterization of Gp65 as a DNA dependent AAA ATPase indicates its role in DNA repair/maintenance mechanism in mycobacteriophages.
“…Recombinase RecA is crucial for DNA repair. RecA bind to ssDNA break and scan for a homologous template dsDNA (103). Miller-Messmer et al (104) studied DNA recombination events in mitochondria.…”
Impatiens is a genus containing more than 1000 species. Thanks to its size, it is a unique system for studying species diversification in natural populations. This study focused on the characterization of novel transcriptomes from seven Impatiens species originating from Nepal. Leave transcriptome of Impatines balsamina L. , I. racemosa DC. , I. bicornuta Wall , I. falcifer Hook , I. devendrae Pusalkar, I. scullyi Hook and I. scabrida DC were sequenced and compared. Reads were de novo assembled and aligned to 92 035-226 081 contigs. We identified 14 728 orthology groups shared among all the species and 3 020 which were unique to a single species. In single species, 2536-3009 orthology groups were under selection from which 767 were common for all species. Six of the seven investigated species shared 77% of gene families with I. bicornuta being the most distinct species. Specific gene families involved in response to different environmental stimuli were closely described. Impatiens bicornuta selection profile shared selection on zing finger protein structures and flowering regulation and stress response proteins with the other investigated species. Overall, the study showed substantial similarity in patterns of selections on transcribed genes across the species suggesting similar evolutionary pressures. This suggests that the species group may have evolved via adaptive radiation.
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