Abortive ligation during base excision repair (BER) leads to blocked repair intermediates containing a 5'-adenylated-deoxyribose phosphate (5'-AMP-dRP) group. Aprataxin (APTX) is able to remove the AMP group allowing repair to proceed. Earlier results had indicated that purified DNA polymerase β (pol β) removes the entire 5'-AMP-dRP group through its lyase activity and flap endonuclease 1 (FEN1) excises the 5'-AMP-dRP group along with one or two nucleotides. Here, using cell extracts from APTX-deficient cell lines, human Ataxia with Oculomotor Apraxia Type 1 (AOA1) and DT40 chicken B cell, we found that pol β and FEN1 enzymatic activities were prominent and strong enough to complement APTX deficiency. In addition, pol β, APTX and FEN1 coordinate with each other in processing of the 5'-adenylated dRP-containing BER intermediate. Finally, other DNA polymerases and a repair factor with dRP lyase activity (pol λ, pol ι, pol θ and Ku70) were found to remove the 5'-adenylated-dRP group from the BER intermediate. However, the activities of these enzymes were weak compared with those of pol β and FEN1.

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.

OBJECTIVE

The local recurrence ratio following surgery alone is higher in patients with rectal cancer than in those with colon cancer. Preoperative radiotherapy reduces the rate of local recurrence and improves the chances of survival in patients with resectable advanced rectal carcinoma. Identification of predictive indicators of radiosensitivity is useful in selecting patients best suited for preoperative radiotherapy and thus helps to avoid unnecessary preoperative treatment. We investigated whether the combination of Ku, p53, p21, and p16 predicted tumor radiosensitivity.

METHODS

We studied 96 cases with advanced rectal carcinoma. In preradiation biopsy specimens of tumor samples, all immunoreactive nuclei of cells stained positive for Ku, p53, p21, and p16 were evaluated by immunohistochemistry. The expression of p53, p21 and p16 in more than 5% of tumor cells were defined as positive, whereas both Ku70 and Ku86 protein in more than 70% of such cells were defined as Ku positive. The expression patterns of Ku, p53, p21, and p16 were examined for association with tumor radiosensitivity, which was determined according to the criteria of histopathologic assessment of radiotherapy effects.

RESULTS

Univariate analysis showed a correlation between the expression patterns of Ku, p53, p21, and p16 and tumor radiosensitivity, while multivariate analysis showed that the expression pattern of Ku and p16 significantly correlated with tumor radiosensitivity. The combination of Ku and p16, or Ku, p53, p21 and p16 was therefore a good predictive marker for tumor radiosensitivity.

CONCLUSIONS

These findings tend to support the hypothesis that the combination of Ku, p53, p21, and p16 expression after radiotherapy can act as a marker for radiosensitivity. Further investigation is needed, as the number of cases in this study was limited.

The identification of predictive indicators of radiosensitivity is extremely useful in selecting patients suited for preoperative radiotherapy and avoiding unnecessary preoperative treatment. In this study, we evaluated the possible role of the immunohistochemical expression pattern of p53 and Ku70 protein in determining tumor radiosensitivity in rectal cancer before preoperative irradiation. We examined pretreatment biopsy materials from 111 patients by immunohistochemistry. The expression pattern of p53 and Ku70 was evaluated for association with tumor radiosensitivity, which was defined according to the criteria of the Japanese Research Society for Cancer of the Colon and Rectum. There was a significant correlation between the expression pattern of p53 and tumor radiosensitivity (P = 0.045); Ku70 and tumor radiosensitivity (P < 0.001); and the combination of p53 and Ku70, and tumor radiosensitivity (P < 0.001). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy in both p53 and Ku70-positive cases for radioresistance were all superior to those of the group positive for p53 alone. In conclusion the examination of the combination of p53 and Ku70 may predict the radiosensitivity of rectal cancer before preoperative irradiation.

BACKGROUND

Activator protein-2 (AP-2) alpha and AP-2gamma transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity.

METHODS

Ku proteins were identified as AP-2alpha interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression.

RESULTS

Nuclear proteins from BT-474 cells overexpressing AP-2alpha and AP-2gamma were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2alpha activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2alpha on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2alpha and AP-2gamma siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2alpha and AP-2gamma or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2alpha and AP-2gamma. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter.

CONCLUSIONS

Ku proteins in interaction with AP-2 (alpha and gamma) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells.

We investigated whether the levels of the DNA-dependent protein kinase (DNA-PK) activity and content correlate with drug sensitivity in different tumour materials and if this can be utilised in predicting treatment outcome. DNA-PK activity and expression were investigated in tumour cells from 8 patients with chronic lymphocytic leukaemia (CLL) and 18 patients with acute myeloid leukaemia (AML), using Western blot and DNA-PK kinase activity assay. Tumour cells from the patients were investigated for their drug sensitivity to topoisomerase II inhibitors (doxorubicin and etoposide), DNA reactive agents (melphalan, 4-hydroxycyclophosphamide and cisplatinum), an antimetabolite (cytosine arabinoside) and an antimicrotubule agent (vincristine) by fluorometric microculture cytotoxicity assay (FMCA). Within each group of leukaemia there was a large variation in both DNA-PK activity and DNA-PKcs expression, while the Ku subunits were expressed more homogeneously. In CLL cells, sensitivity to topoisomerase II inhibitors correlated with DNA-PKcs protein expression (r=0.7174, p=0.0452). In AML samples, sensitivity to DNA cross-linking alkylating agents correlated with Ku86 (r=-0.7512, p=0.0031) and Ku70 (r=-0.6134, p=0.0258) expression. Unexpectedly, DNA-PK activity was found to correlate with sensitivity to vincristine in both CLL (r=0.8557, p=0.0067) and AML (r=0.5480, p=0.0228) cells. The results indicate that DNA-PK is not only involved in the recognition of DNA double-strand breaks (DSB), but also other DNA lesions.

Ku70 is a protein that finds itself at the heart of several important cellular processes. It is essential to the non-homologous end joining pathway as a part of the DNA-end binding complex, required for proper maintenance of telomeres and contributes to DNA damage recognition and regulation of apoptosis. Forces that regulate Ku70 are therefore likely to have large consequences on the physiologic state of the cell. We report here that transient expression of the small protein SUMO resulted in a dramatic increase in the abundance of Ku70. Surprisingly, the direct SUMOylation of Ku70 does not appear to be required for this effect. Rather, Ku70 appears to be stabilized through indirect effects on the rate of degradation. The same outcome was obtained by raising the expression of enzymes that promote SUMOylation. It is likely that many other proteins will be similarly regulated, providing a general control of cellular state.

Bloom's syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3'-5' DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA double-strand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report first that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcs-dependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, reflecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.

The Ku70 protein was shown to be involved in multiple cellular pathways including DNA repair, telomere maintenance, V(D)J recombination and Bax mediated apoptosis. Yet, despite this wide spectrum of pathways, until recently the enzymatic activity of Ku70 was elusive. Recent findings demonstrate that Ku70 is associated with the proapoptotic protein Bax and possesses a deubiquitin enzyme (DUB) activity on it. These data suggest a dual role for Ku70 in apoptotic regulation; on one hand the association with Ku70 sequestered Bax away from the mitochondria and plays an antiapoptotic function. On the other hand, this association mediates and promotes Bax deubiquitylation which will block its labeling for proteasomal degradation and in this manner Ku70 will have a proapoptotic role. The exciting finding of Ku70's DUB activity opens numerous avenues for future research. Here we suggest candidate substrate proteins and indicate how the DUB activity of Ku70 on these, might affect the known Ku70's related pathways.

Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs alone, or in combination with other factors, to introduce a point mutation into the alphaB-crystallin gene in one-celled mouse embryos. We found that co-injection of ssDNA along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative nonhomologous end-joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been previously reported.

The autoantigen Ku, composed of subunits Ku70 and Ku86, is necessary for repair of DNA double-strand breaks by nonhomologous end joining. Similarly, Ku participates in repair of DNA double-strand breaks that occur during V(D)J recombination, and it is therefore required for the development of B and T lymphocytes. Although previous studies have identified the DNA-binding activities of Ku, little is known concerning its dynamics, such as the mobility of Ku in the nucleus and its rate of association with substrates. To address this question, fluorescence photobleaching experiments were performed using HeLa cells and B cells expressing a green fluorescent protein (GFP) fusion construct of either Ku70 or Ku86. The results show that Ku moves rapidly throughout the nucleus even following irradiation of the cells. However, the rate of diffusion of Ku was approximately 100-fold slower than that predicted from its size. Association of Ku-GFP with a filamentous nuclear structure was also evident, and nuclear extraction experiments suggest that this represents nuclear matrix. A central domain of Ku70 containing its DNA-binding and heterodimerization regions and its nuclear localization signal shows that this alone is sufficient for the observed mobility of Ku70-GFP and its association with nuclear matrix. These data suggest the mobility of Ku is characterized by a transient, high flux association with nuclear substrates that includes both DNA and the nuclear matrix and may represent a mechanism for repair of double-strand breaks using the nuclear matrix as a scaffold.

The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg(2+), implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.

Hypoxia may inhibits the NHEJ DNA repair through downregulating Ku70/80 expression and combined with an increased angiogenesis and altered p53 expression would be responsible for tumor progression in cervical carcinoma.

In somatic cells, eroded telomeres can induce DNA double-strand break signaling, leading to a form of replicative senescence or apoptosis, both of which are barriers to tumorigenesis. However, cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis, enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction, and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3' single-stranded overhangs and an increased number of telomere-induced foci containing gammaH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs, 2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably, these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions, or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together, our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction.

OBJECTIVE

The DNA repair gene Ku70, an important member of non-homologous end-joining repair system, is thought to play an important role in the repairing of DNA double strand breaks. It is known that defects in double strand break repair capacity can lead to irreversible genomic instability. However, the polymorphic variants of Ku70, have never been reported about their association with gastric cancer susceptibility.

METHODS

In this hospital-based case-control study, the associations of Ku70 promoter T-991C (rs5751129), promoter G-57C (rs2267437), promoter A-31G (rs132770), and intron 3 (rs132774) polymorphisms with gastric cancer risk in a Taiwanese population were investigated. In total, 136 patients with gastric cancer and 560 age- and gender-matched healthy controls recruited from the China Medical Hospital in Taiwan were genotyped.

RESULTS

As for Ku70 promoter T-991C, the ORs after adjusted by age and gender of the people carrying TC and CC genotypes were 2.41 (95% CI = 1.53-3.88) and 3.21 (95% CI = 0.96-9.41) respectively, compared to those carrying TT wild-type genotype. The P for trend was significant (P < 0.0001). In the dominant model (TC plus CC versus TT), the association between Ku70 promoter T-991C polymorphism and the risk for gastric cancer was also significant (adjusted OR = 2.48, 95% CI = 1.74-3.92). When stratified by age and gender, the association was restricted to those at the age of 55 or elder of age (TC vs TT: adjusted OR = 2.52, 95% CI = 1.37-4.68, P = 0.0139) and male (TC vs TT: adjusted OR = 2.58, 95% CI = 1.33-4.47, P = 0.0085). As for the other three polymorphisms, there was no difference between both groups in the distributions of their genotype frequencies.

CONCLUSIONS

In conclusion, the Ku70 promoter T-991C (rs5751129), but not the Ku70 promoter C-57G (rs2267437), promoter A-31G (rs132770) or intron 3 (rs132774), is associated with gastric cancer susceptibility. This polymorphism may be a novel useful marker for gastric carcinogenesis.

Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID)-dependent hypermutation of Ig V(D)J rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(D)J regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(D)J hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs.

Variation in DNA repair genes is one of the mechanisms that may lead to variation in DNA repair capacity. Ku, a heterodimeric DNA-binding complex, is directly involved in repair of DNA double-strand breaks. Ku consists of two subunits, Ku70 and Ku80, which are encoded by the XRCC6 and XRCC5 genes, respectively. In the present study, we investigated whether common genetic variant in variable number of tandem repeats (VNTR) XRCC5 and T-991C XRCC6 was associated with an altered risk of breast cancer. The present study included 407 females with breast cancer and 395 age frequency-matched controls which were randomly selected from the healthy female blood donors. The XRCC5 and XRCC6 polymorphisms were determined using PCR-based methods. For XRCC5 polymorphism, in comparison with the 1R/1R genotype, the 0R/0R genotype increased breast cancer risk (OR 9.55, 95%CI 1.19-76.64, P = 0.034). The 1R/3R genotype compared with 1R/1R genotype decreased the risk of breast cancer (Fisher's exact test P = 0.015). There was no association between T-991C polymorphism of XRCC6 and breast cancer risk. Mean of age at diagnosis of breast cancer for 0, 1, 2, 3, and >4 repeat in XRCC5 were 39.2, 41.9, 44.3, 45.8, and 47.3 years, respectively. The Kaplan-Meier survival analysis revealed that the number of repeat was associated with age at diagnosis of breast cancer (log rank statistic = 13.90, df = 4, P = 0.008). The findings of the present study revealed that either breast cancer risk or age at diagnosis of breast cancer was associated with the VNTR polymorphism at promoter region of XRCC5.

In order to investigate the molecular basis of variation in response to ionising radiation (IR) in radiotherapy patients, we have studied the expression of several genes involved in DNA double-strand break repair pathways in fibroblast cell lines. Ten lines were established from skin biopsies of cancer patients with different normal-tissue reactions to IR, and 3 from a control individual. For all 10 test cell lines, the cellular radiosensitivity was also known. Using Western blots we measured, in non-irradiated cells, the basal expression levels of ATM, Rad1 and Hus1, involved in the control of cellular DNA damage checkpoints, together with DNA-PKcs, Ku70, Ku80; XRCC4, ligaseIV and Rad51, involved in radiation- induced DSB repair. We also analysed the in vitro enzymatic activities, under non-irradiated conditions, of the DNA-PK and XRCC4/ligaseIV complexes. The levels of expression of the different proteins were similar in all the cell lines, but the activities of the DNA-PK and XRCC4/ligaseIV complexes showed some differences. These differences did not correlate with either the normal tissue response of the patient in vivo or with cellular radiation sensitivity in vitro. The activity differences of these enzyme complexes, therefore, do not account for the variation of responses seen between patients.

We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70(+) strain due to the low frequency of homologous recombination were efficiently obtained from Delta ku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.

DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V(D)J recombination, class switch recombination and telomere maintenance.

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

LKB1/STK11 is a tumor suppressor gene responsible for Peutz-Jeghers syndrome, an inherited cancer disorder associated with genome instability. The LKB1 protein functions in the regulation of cell proliferation, polarization and differentiation. Here, we suggest a role of LKB1 in non-homologous end joining (NHEJ), a major DNA double-strand break (DSB) repair pathway. LKB1 localized to DNA ends upon the generation of micro-irradiation and I-SceI endonuclease-induced DSBs. LKB1 inactivation either by RNA interference or by kinase-dead mutation compromised NHEJ-mediated DNA repair by suppressing the accumulation of BRM, a catalytic subunit of the SWI/SNF complex, at DSB sites, which promotes the recruitment of an essential NHEJ factor, KU70. AMPK2, a major substrate of LKB1 and a histone H2B kinase, was recruited to DSBs in an LKB1-dependent manner. AMPK2 depletion and a mutation of H2B that disrupted the AMPK2 phoshorylation site impaired KU70 and BRM recruitment to DSB sites. LKB1 depletion induced the formation of chromosome breaks and radials. These results suggest that LKB1-AMPK signaling controls NHEJ and contributes to genome stability.