Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The VSports app下载. gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2016 May:41:16-26.
doi: 10.1016/j.dnarep.2016.03.003. Epub 2016 Mar 24.

Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair

Mohammed Almohaini  1 , Sri Lakshmi Chalasani  1 , Duaa Bafail  1 , Konstantin Akopiants  1 , Tong Zhou  1 , Steven M Yannone  2 , Dale A Ramsden  3 , Matthew C T Hartman  4 , Lawrence F Povirk  5
Affiliations

Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair

Mohammed Almohaini et al. DNA Repair (Amst). 2016 May.

Abstract (V体育安卓版)

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends VSports手机版. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined. .

PubMed Disclaimer

Figures

Figure 1
Figure 1
Tg-containing DSB substrates. A. Construction of modified substrates from short, end-labeled (*) Tg-containing duplexes and a fragment of pUC19. B. Terminal structures and sequences of the substrates. C. Formation of head-to-tail and head-to-head end joining products, and their detection as fragments of NdeI/PstI cleavage.
Figure 2
Figure 2
Effect of Tg on joining of blunt-ended substrates by NHEJ. The indicated site-specifically labeled substrates, either unmodified or containing Tg at the first, second or third position from the terminus of the labeled end, were incubated in XLF-deficient Bustel extracts (or heat-inactivated extracts), supplemented with XLF (100 nM), Artemis (80 nM), and/or ddTTP in place of dTTP as indicated, for 6 hr at 37°C. The samples were deproteinized, in some cases treated with EndoIII, then cut with NdeI and PstI and analyzed on denaturing gels. Lanes marked “M” contain 5′-end-labeled 36- and 44-base oligomers of the sequence expected for blunt-end ligation products. Bar graphs show yield of specific products of the indicated substrates and error bars indicate mean ± SEM for 4 replicate experiments.
Figure 3
Figure 3
Presence of Tg in end joining products. Tg-containing or unmodified substrates were incubated for 6 hr in Bustel extracts supplemented with XLF and ddTTP as indicated. Samples were deproteinized and cut with NdeI and PstI, then denatured and annealed to 44-base complements of the expected head-to-tail ligation products and treated (or not) with EndoIII prior to denaturing gel electrophoresis. Bar graphs show the yield of 44-base products in each case (mean ± SEM for 3 independent experiments).
Figure 4
Figure 4
Time course for Tg3 and Tg1 end joining and effect of dideoxynucleotides. A. and B. Tg3 was incubated in extracts containing ddTTP, ddCTP and/or XLF for the times indicated, then cut with NdeI and PstI and analyzed as in Fig. 2. One sample in (A.) was treated with EndoIII prior to NdeI/PstI cleavage, as in Fig. 2. Asterisk (*) indicates addition of a mutant XLF with an L115A mutation. C. Quantitative analysis of Tg3 ligation in the presence of dNTPs, derived from three replicates of the experiment shown in (B.). D. Time course of formation of end joining for Tg1. The Tg1 substrate was incubated in extracts, with XLF added either at the start of the reaction (●) or after 2 hr incubation (□). Reaction conditions were as in Fig. 2.
Figure 5
Figure 5
Interference between BER and end joining of Tg5. A. Either the Tg5 substrate or a corresponding unmodified substrate was incubated in extracts containing XLF for the times indicated, then cut with NdeI and PstI and analyzed as in Fig. 2. B. Quantitative analysis of three experiments with the Tg5 substrate, showing levels of the truncated 14-base fragment or the 44-base head-to-tail end joining product. C. Verification of a recessed 3′-hydroxyl terminus. The Tg5 substrate was incubated in extract for the indicated times, then treated with exonuclease-deficient Klenow fragment prior to NdeI/PstI cleavage. No Ext. = incubation in heat-inactivated extract for 6 hr.
Figure 6
Figure 6
Tg cleavage by purified hNTH. A. The Tg5 substrate was treated with the indicated concentrations of hNTH for 1 hr, then cut with NdeI prior to denaturing gel electrophoresis. Sample in leftmost lane was also treated with TDP1 to remove 3′-dRp. B. Tg cleavage as a function of hNTH concentration for the Tg3 and Tg5 substrates (mean ± SEM for 3 experiments). The Tg1 and Tg2 substrates showed no detectable cleavage at any hNTH concentration.
Figure 7
Figure 7
Effect of 3′-proximal Tg on end joining in HCT116 extracts. The indicated substrates were inbubated in unsupplemented extracts of HCT116 cells and end joining was analyzed as in Fig. 2. A. Gel electrophoresis of end joining products. B. Pooled data from three replicate experiments showing abundance of the head-to-tail 44-bp product.
Figure 8
Figure 8
Ligation of Tg-containing substrates by purified Ku, X4L4 and XLF. A. The indicated substrates were incubated with 10 nM Ku, 40 nM X4L4 and 50 or 100 nM XLF as indicated for 4 hr, then deproteinized and cut with NdeI and PstI and analyzed on a sequencing gel. B. Quantitation of results from 3 independent experiments with 100 nM XLF (mean ± SEM).
Figure 9
Figure 9
Effect of 5′-proximal Tg on NHEJ. The blunt-ended substrates shown, with Tg as the second or third base from the 5′ terminus, were incubated for 6 hr in XLF-complemented Bustel extracts. A. Substrates and head-to-tail ligation products. The palindromic 36-base head-to-head product is identical to that shown in Fig. 1C, except for terminal sequence. B. Denaturing gel analysis of end joining products following cleavage with NdeI and PstI. C. Pooled data for formation of head-to-tail products, from 3 independent experiments (mean ± SEM). D. Cleavage of 5′-proximal Tg substrates by purified hNTH (mean ± SEM for 3 experiments). Substrates were incubated with the indicated concentrations of hNTH, and release of the 5′-terminal 1- or 2-base fragment was assessed by gel electrophoresis (see Supplemental Fig. 4).
See this image and copyright information in PMC

References

    1. Hall EJ, Giaccia AJ. Radiobiology for the Radiologist Wolters Kluwer/Lippincott, Williams & Wilkins. 2012.
    1. Ward JF. Biochemistry of DNA lesions. Radiat Res Suppl. 1985;8:S103–11. - PubMed
    1. Goodhead DT. Initial events in the cellular effects of ionizing radiations: Clustered damage in DNA. Int J Radiat Biol. 1994;65:7–17. - PubMed
    1. Georgakilas AG, O’Neill P, Stewart RD. Induction and repair of clustered DNA lesions: What do we know so far? Radiat Res. 2013;180:100–109. - PubMed
    1. Shibata A, Conrad S, Birraux J, Geuting V, Barton O, Ismail A, Kakarougkas A, Meek K, Taucher-Scholz G, Lobrich M, Jeggo PA. Factors determining DNA double-strand break repair pathway choice in G2 phase. Embo j. 2011;30:1079–1092. - PMC - PubMed

Publication types

Substances

LinkOut - more resources