The repair of DSBs can be performed by the NHEJ pathway that has been widely characterized in in vitro
assays utilizing restriction endonuclease generated DSBs with compatible or mismatched ends. Several recent studies have indicated that, in the absence of functional classical NHEJ, the repair of DSBs containing mismatched overhangs can be achieved by the extensive end-processing of DNA to produce ligatable ends. Such pathways have been referred to as alternative NHEJ (16
), microhomology-based single strand annealing (21
) and microhomology-mediated end-joining (23
) and are speculated to be mutagenic due to the loss of genomic sequence.
In this study we have measured the DSB end-joining capacity of high grade bladder tumours which are associated with high levels of genomic instability. We have used cell-free extract from the MO59K human glioblastoma cell line to define the type of DSB end-joining expected in an NHEJ proficient cell and then compared this with the end-joining in extracts prepared from four high grade bladder tumours and five normal urothelial samples. Repair of compatible and partially incompatible DSBs by MO59K was accurate however, DSBs with fully mismatched overhangs could be joined by the non-random deletion of terminal nucleotides at regions of microhomology indicating the presence of an error-prone end-joining mechanism constitutively present at a low level in normal cells. Use of the partially incompatible DSB substrate I2 revealed that while the NHU extracts produced mostly accurate joins, bladder tumour extracts joined with lower fidelity and utilized regions of microhomology within the DNA substrate in a manner that was not dependent on Ku, DNA-PK or XRCC4 and strikingly similar to joins formed in extracts in which Ku70 or DNA-PKcs were not active.
How could DSBs be joined in the absence of classical NHEJ? The joining of DSBs in NHEJ proficient cells has been shown to result in the formation of predominantly accurate joins, where DNA-PK holoenzyme is predicted to stabilize the association of short mismatched overhangs by single nucleotide interactions so allowing fill-in synthesis and ligation to occur (18
). In addition the ligase IV/XRCC4 complex also plays a role in protecting DNA from end-degradation so promoting accurate joining (25
). In the absence of classical NHEJ the association of incompatible DNA ends would have to involve the base-pairing of DNA sequences via regions of microhomology within the DNA molecule such that the deletion of non-base-pairing terminal nucleotides would be necessary prior to ligation. The joining of mismatched ends in this way would require the search and recognition of regions of microhomology and resection of DNA substrate by exonucleolytic activity to form ends which would be ligatable. It is possible that bladder tumour extracts could contain increased levels of non-specific nucleases which would result in the random deletion of terminal nucleotides from the DSB; however, the non-random, asymmetric end-processing and use of microhomology by these extracts, and also in extracts in which ether Ku or DNA-PKcs
are inactive, points to the involvement of a regulated exonuclease activity. The M/R/N complex contains independent alignment and nuclease functions ideal for DSB processing (39
) and, as junctions in this study are formed between sequences found near or at one end of the DSB and those buried some distance from the other end, M/R/N could be involved in the detection and alignment DNA microhomologies prior to resection of 3′ overhangs by an as yet unidentified nuclease (40
The reduced joining fidelity observed in bladder tumours could be due to a deficiency for a critical NHEJ factor or increased expression of proteins involved in the error-prone pathway. Western blot analysis of bladder tumour extracts did not identify a common deficiency in NHEJ components or an increase in M/R/N levels that would account for the predominance of error-prone joining, although there was a generalized reduction in the levels of NHEJ components in both NHUs and bladder tumours. The full length Nbs1 protein observed in bladder tumour extracts was accompanied by a smaller immunoreactive protein not apparent in MO59K or NHU. Truncated Nbs-1 has been observed in patients with Nijmegen breakage syndrome and results from translation from an internal initiation site such that the N-terminal portion of the protein is not expressed (42
). However, as N-terminal truncated Nbs1 retains Mre11 and Rad50 binding capacity and this region of the protein is dispensable for DSB repair and survival it seems unlikely that this will impact on the joining fidelity observed in this study (43
). Truncation of Mre11 was observed in BT1 the extract that was associated with a particularly high level of error-prone joining of complementary DSBs. A similarly sized truncated Mre11 was observed in the chromosomal instability syndrome AT-like disorder, which predisposes to cancer, and results from a point mutation causing premature termination of Mre11 at the C-terminal (44
) and loss of critical C-terminal DNA binding domains (43
It is possible that whilst the levels of NHEJ components are not altered in bladder tumours compared with NHU the activity of these proteins could be altered. Reduced DNA binding by Ku has been observed in aggressive basal cell carcinoma biopsies compared with normal controls (45
). In a separate study the Ku-binding characteristics of seven bladder tumours were measured by gel retardation; five low grade tumours (grade 1–2, pTa–T1) displayed increased DNA binding by Ku whilst two high grade invasive tumours (grade 3, pT3) had a 1.5- to 3-fold decrease (46
). The ability of Ku to interact with DNA ends could be altered in the high grade bladder tumours in this study. Similarly, alterations in the activity of DNA-PKcs
, the DNA-PK holoenzyme or ligase IV/XRCC4 complex could also affect DNA end-protection and end-joining fidelity. Interestingly, in V(D)J recombination the RAG-associated post-cleavage complex is thought to guide the bound DNA ends towards classical NHEJ so avoiding aberrant joining by alternative end-joining processes (47
). Similar ‘shepherding’ roles for proteins may exist in a non-V(D)J context to ensure that repair of DSBs is undertaken by accurate NHEJ; alterations in proteins involved in this role could leave broken DNA ends open to repair by a usually minor, less faithful pathway (47
). Therefore, in bladder tumour extracts alterations in the function of proteins involved in classical NHEJ could shift the balance away from NHEJ and towards error-prone microhomology-mediated end-joining.
Genomic instability is a hallmark of cancer and the accurate repair of DSBs is essential for maintenance of chromosome integrity. Compromised NHEJ can be compensated for, to a certain extent, by HR (48
) and remaining cells containing unrepaired breaks are most likely eliminated by apoptosis (50
). Loss of p53 function in a Ku null background enhances chromosomal instability, increases joining of translocation breakpoints at regions of microhomology and promotes tumour formation in mice (37
). Bladder tumours have been found to carry inactivating mutations in p53 or Rb and a large number of genomic deletions/amplifications (26
). In this study we show that, bladder tumours utilize error-prone end-joining in preference to accurate NHEJ indicating a deficiency in the classical NHEJ pathway. This highly mutagenic joining of DSBs via sequence microhomologies may contribute to the increased genomic instability observed in high grade bladder cancer.