Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • br DNA end processing enzymes The simplest DSB

    2020-02-06


    DNA end processing enzymes The simplest DSB is one that consists of two blunt DNA ends as these termini can be re-joined without processing. However, DSBs induced by ionizing radiation and reactive oxygen species are notorious for producing DNA ends which are non-ligatable (“dirty ends”) and thus must be processed in order for ligation. Depending on the nature of the DNA break, different DNA end processing enzymes may be required, including those that resect DNA ends, fill in gaps, and remove blocking end groups, to create DNA ends that are ligatable. The long list of enzymes that the NHEJ pathway employs to process DNA ends underscores the versatility of the pathway. How specific enzymes are correctly chosen to fix each “dirty” end is not completely understood. Ku70/80 recruits, along with the canonical NHEJ factors, a large number of these processing enzymes to the DSB, which has resulted in Ku being called a “tool-belt” protein [107]. The thought is that Ku recruits and stabilizes NHEJ factors which it deems required for the repair of each specific DSB [108]. This is supported by a study showing that the complexity of DNA damage influences the recruitment of NHEJ factors to the DSB [109]. Simple DSBs can be rapidly repaired by Ku70/80, XRCC4, R788 disodium mg IV, and XLF and complex breaks requiring those factors along with DNA-PKcs and possibly the activity of ATM. Collectively, this suggests the Ku heterodimer likely directs the utilization of specific subcomplexes to guide repair of individual DSBs. However, there is some belief that a fraction of NHEJ proceeds by a “trial and error” process in which each processing enzyme has an opportunity to fix the dirty end and that this process continues until the DSB is ligated and thus repaired [108]. A common incompatible end is one that contains a 3′ or 5′ single-strand overhang. These overhangs can be removed either by direct resection of the overhang or by using the nucleotide sequence of the overhang as a template to duplicate a complementary strand. The proteins implicated in resecting DNA ends for NHEJ include the endonucleases Artemis and Metnase, the RecQ helicase family member WRN, and aprataxin and PNKP-like factor (APLF). Artemis is the nuclease with the best established requirement for NHEJ. The confirmed activities of Artemis include a 5′ endonucleases activity with a preference to nick a 5′ overhang which leaves a blunt duplex end and the ability to remove 3′-phosphoglycolate groups from DNA termini [110], [111]. It should be noted that tyrosyl-DNA phosphodiesterase (Tdp1) can also repair 3′-phosphoglycolate-terminated DSBs and likely operates in NHEJ on special DSBs [112]. As stated above, the endonucleolytic activity of Artemis is regulated by DNA-PKcs and its phosphorylation status at the Thr2609 cluster [110]. Furthermore, Artemis can nick regions ssDNA within gaps in the double-stranded DNA and regions of mismatched bases which may be required for NHEJ [113]. Lastly, Artemis is required for the repair of complex DSBs and those that occur in the heterochromatin [114]. Metnase is an endonuclease that preferentially cleaves ssDNA and ssDNA-overhang of a partial duplex DNA, but Metnase appears to be not as efficient as Artemis in resolving DNA damaged ends in the presence of the NHEJ machinery [115], [116]. WRN and APLF have both been implicated in the NHEJ pathway. Both proteins have 3′–5′ exonuclease activity in vitro, but the activities of these two enzymes for processing DNA ends for NHEJ in vivo is still not clear [117], [118], [119], [120]. The synthesizing of a complementary strand by adding nucleotides or filling of gaps of DNA for NHEJ is performed by the family X polymerases, which include the polymerases μ (Pol μ) and λ (Pol λ) and terminal deoxynucleotidyl transfer (TdT) [121]. These polymerases display a gradient of template dependency with TdT performing template-independent synthesis, Pol λ is nearly exclusively template-dependent, and Pol μ can carry out both [107], [121], [122]. These polymerases have no proofreading capability and are thus error-prone, but the gap filling activity of Pol λ is accurate in the presence of Ku and XRCC4/DNA ligase IV [123]. Pol λ is tolerant of base damage, as it has been shown to have lyase activity [124]. Pol μ can catalyze DNA in the absence of complementary ends of the DNA via a unique end-bridging activity [125]. TdT is only found in lymphocytes and thus only plays a role in NHEJ during V(D)J recombination [126].