iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://www.ncbi.nlm.nih.gov/pubmed/15692565?dopt=Abstract
Processing of DNA for nonhomologous end-joining by cell-free extract - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .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.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Feb 23;24(4):849-60.
doi: 10.1038/sj.emboj.7600563. Epub 2005 Feb 3.

Processing of DNA for nonhomologous end-joining by cell-free extract

Joe Budman  1 , Gilbert Chu
Affiliations

Processing of DNA for nonhomologous end-joining by cell-free extract

Joe Budman et al. EMBO J. .

Abstract

In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. We have developed a cell-free system capable of processing and joining noncompatible DNA ends. The system had key features of NHEJ in vivo, including dependence on Ku, DNA-PKcs, and XRCC4/Ligase4. The NHEJ reaction had striking properties. Processing of noncompatible ends involved polymerase and nuclease activities that often stabilized the alignment of opposing ends by base pairing. To achieve this, polymerase activity efficiently synthesized DNA across discontinuities in the template strand, and nuclease activity removed a limited number of nucleotides back to regions of microhomology. Processing was suppressed for DNA ends that could be ligated directly, biasing the reaction to preserve DNA sequence and maintain genomic integrity. DNA sequence internal to the ends influenced the spectrum of processing events for noncompatible ends. Furthermore, internal DNA sequence strongly influenced joining efficiency, even in the absence of processing. These results support a model in which DNA-PKcs plays a central role in regulating the processing of ends for NHEJ.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Assay for NHEJ in vitro. DNA substrates were synthesized with various DNA ends attached to the same internal DNA sequences, DNA1 and DNA2. The figure shows substrates with compatible and noncompatible ends. In some cases, we synthesized DNA substrates in which the DNA ends were exchanged with respect to the internal DNA sequences, as illustrated for the noncompatible ends in the figure. Joining efficiency was measured by qPCR. Processing of the ends was analyzed after amplifying DNA junctions by PCR and sequencing individual clones.
Figure 2
Figure 2
Extract supports NHEJ of noncompatible ends. (A) NHEJ is detectable by a gel-based assay. In lanes 2, 6, 10, and 14, DNA substrates with compatible or noncompatible ends were incubated with extract and dNTPs. The resulting junctions were amplified by 30 cycles of PCR, and the PCR product was resolved by gel electrophoresis. In lanes 3, 7, 11, and 15, the extract was pre-incubated for 30 min with 10 μM wortmannin (wort) to inhibit the kinase activity of DNA-PKcs. In lanes 4, 8, 12, and 16, end-joining was catalyzed by T4 DNA ligase, which joins only compatible DNA ends. (B) The NHEJ reaction requires DNA-PKcs and XRCC4, while dNTPs stimulate joining of noncompatible ends. DNA substrates with compatible or noncompatible ends were incubated with extract (+dNTPs), extract (−dNTPs), extract (+dNTPs) treated with wortmannin, or extract (+dNTPs) immunodepleted with αXRCC4 antibody (2 × 4). Joining efficiency was measured by qPCR and displayed on a log plot to account for the large variation in joining. The small arrows in place of the bottom two bars represent joining efficiency of less than 0.0001%.
Figure 3
Figure 3
Compatible ends are joined with minimal processing. DNA substrates containing compatible ends were incubated with (A) extract plus added dNTPs, or (B) extract in the absence of added dNTPs. The legends indicate joining efficiency to the left of the arrow, and total number of sequenced junctions to the right of the arrow. The number of times each junction was recovered is shown in the column labeled ‘No'. When the DNA ends are processed, the open-mouthed icon and white background in the DNA indicate nuclease activity, and the arrow and black background indicate polymerase activity. Gray background indicates unprocessed DNA.
Figure 4
Figure 4
In the presence of dNTPs, noncompatible ends are processed largely by templated nucleotide addition. DNA substrates with compatible or noncompatible ends were incubated with extract and dNTPs, and the junctions were sequenced. The processed ends are shown according to the legend in Figure 3. The number of bases of microhomology utilized in the joining reaction is shown in the column labeled ‘MH'. Each section (A–E) depicts junctions from a different structural category of paired ends.
Figure 5ab
Figure 5ab
When dNTPs are omitted, noncompatible ends are processed by deletion and templated addition of nucleotides. DNA substrates with noncompatible ends were incubated with extract in the absence of added dNTPs, and the processed ends are shown according to the legend in Figure 3. Asterisks indicate junctions that utilized microhomology from internal DNA sequences. In these cases, there was ambiguity in the processing events, and we assumed that the microhomology alignment occurred via a 3′ overhang. Each section (A–E) depicts junctions from a different structural category of paired ends.
Figure 5cde
Figure 5cde
When dNTPs are omitted, noncompatible ends are processed by deletion and templated addition of nucleotides. DNA substrates with noncompatible ends were incubated with extract in the absence of added dNTPs, and the processed ends are shown according to the legend in Figure 3. Asterisks indicate junctions that utilized microhomology from internal DNA sequences. In these cases, there was ambiguity in the processing events, and we assumed that the microhomology alignment occurred via a 3′ overhang. Each section (A–E) depicts junctions from a different structural category of paired ends.
Figure 6
Figure 6
Joining efficiency of blunt DNA ends is influenced by internal DNA sequence. Joining efficiency was measured after incubating DNA substrates with T4 DNA ligase, or after incubating the same DNA substrates with extract. In these experiments, the reactions were performed in the absence of added dNTPs.
Figure 7
Figure 7
Model for NHEJ. Ku binds to DNA ends and recruits DNA-PKcs, which mediates synapsis of the ends. XRCC4/Ligase4, polymerase, Artemis, and perhaps another nuclease assemble at the synaptic complex. Threading of single-stranded DNA ends into cavities in the DNA-PKcs molecule activates the kinase. DNA-PKcs phosphorylates Artemis, activating its endonuclease activity. DNA-PKcs undergoes autophosphorylation and moves away from the DNA ends. This may provide preferential access to XRCC4/Ligase4. If the ends are compatible, ligation occurs immediately. If the ends are not compatible, XRCC4/Ligase4 remains in the synaptic complex, while polymerase and nuclease activities process the ends. As soon as the ends are processed into a compatible substrate, XRCC4/Ligase4 completes the joining reaction.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baumann P, West SC (1998) DNA end-joining catalyzed by human cell-free extracts. Proc Natl Acad Sci USA 95: 14066–14070 - PMC - PubMed
    1. Bebenek K, Garcia-Diaz M, Blanco L, Kunkel TA (2003) The frameshift infidelity of human DNA polymerase lambda. Implications for function. J Biol Chem 278: 34685–34690 - PubMed
    1. Boe SO, Sodroski J, Helland DE, Farnet CM (1995) DNA end-joining in extracts from human cells. Biochem Biophys Res Commun 215: 987–993 - PubMed
    1. Chan DW, Lees-Miller SP (1996) The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit. J Biol Chem 271: 8936–8941 - PubMed
    1. Chappell C, Hanakahi LA, Karimi-Busheri F, Weinfeld M, West SC (2002) Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining. EMBO J 21: 2827–2832 - PMC - PubMed

Publication types