ABSTRACT
Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that increases the repertoire of antigen receptors by adding non-templated nucleotides (N-addition) to V(D)J recombination junctions. Despite extensive in vitro studies on TdT catalytic activity, the partners of TdT that enable N-addition remain to be defined. Using an intrachromosomal substrate, we show here that, in Chinese hamter ovary (CHO) cells, ectopic expression of TdT efficiently promotes N-additions at the junction of chromosomal double-strand breaks (DSBs) generated by the meganuclease I-SceI and that the size of the N-additions is comparable with that at V(D)J junctions. Importantly, no N-addition was observed in KU80- or XRCC4-deficient cells. These data show that, in a chromosomal context of non-lymphoid cells, TdT is actually able to promote N-addition at non-V(D)J DSBs, through a process that strictly requires the components of the canonical non-homologous end-joining pathway, KU80 and XRCC4.
Subject(s)
Antigens, Nuclear/physiology , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA Nucleotidylexotransferase/metabolism , DNA-Binding Proteins/physiology , Animals , CHO Cells , Cricetinae , Cricetulus , Ku Autoantigen , Nucleotides/metabolism , V(D)J RecombinationABSTRACT
Posttranslational modification of PCNA by ubiquitin plays an important role in coordinating the processes of DNA damage tolerance during DNA replication. The monoubiquitination of PCNA was shown to facilitate the switch between the replicative DNA polymerase with the low-fidelity polymerase eta (η) to bypass UV-induced DNA lesions during replication. Here, we show that in response to oxidative stress, PCNA becomes transiently monoubiquitinated in an S phase- and USP1-independent manner. Moreover, Polη interacts with mUb-PCNA at sites of oxidative DNA damage via its PCNA-binding and ubiquitin-binding motifs. Strikingly, while functional base excision repair is not required for this modification of PCNA or Polη recruitment to chromatin, the presence of hMsh2-hMsh6 is indispensable. Our findings highlight an alternative pathway in response to oxidative DNA damage that may coordinate the removal of oxidatively induced clustered DNA lesions and could explain the high levels of oxidized DNA lesions in MSH2-deficient cells.
Subject(s)
DNA Damage , DNA-Binding Proteins/physiology , DNA-Directed DNA Polymerase/physiology , MutS Homolog 2 Protein/physiology , Oxidative Stress , Proliferating Cell Nuclear Antigen/physiology , Arabidopsis Proteins , Cell Line , Chromatin/metabolism , DNA Polymerase beta/metabolism , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Endopeptidases/metabolism , Humans , MutS Homolog 2 Protein/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Ubiquitin-Specific Proteases , Ubiquitination , X-ray Repair Cross Complementing Protein 1ABSTRACT
Xeroderma pigmentosum (XP) is a group of rare inherited human neurocutaneous diseases, and the group C (XPC) is the major group of patients with XP in Europe, North America, and South America. Current molecular diagnostic methods for XP require specialized, expensive, and time-consuming UV sensitivity and DNA repair assays followed by gene sequencing. To determine whether immunohistochemistry (IHC) would be a robust alternative method to diagnose patients with XPC, we stained sections of paraffin-embedded skin biopsies for XPC by IHC, using 69 archived blocks from confirmed or clinically suspect patients with XPA, XPC, XPD, XPE, and without XP. We found that XPC expression was strong in all skin biopsies from patients without (14 of 14) and other patients with XP (4 of 4), whereas XPC expression was lost in all biopsies from confirmed XPC patients (29 of 29). Patches of strong XPC signal could be detected in sun-damaged skin, squamous and basal cell carcinomas from patients with XPC that colocalized with strong expression of p53 and Ki-67. Patients with XPC can therefore be diagnosed by IHC from paraffin-embedded skin biopsies from regions of skin that are without sun damage or sun-induced tumors. IHC is therefore a robust alternative method to diagnose patients with XPC. This fast and inexpensive method should increase the options for the diagnosis of patients with XPC from paraffin-embedded skin biopsies and could be developed for other complementation groups.
Subject(s)
Immunohistochemistry/methods , Xeroderma Pigmentosum/classification , Xeroderma Pigmentosum/diagnosis , Biopsy , DNA-Binding Proteins/metabolism , Humans , Ki-67 Antigen/metabolism , Lymphocytes/metabolism , Lymphocytes/pathology , Paraffin Embedding , Skin/metabolism , Skin/pathology , Tumor Suppressor Protein p53/metabolism , Xeroderma Pigmentosum/pathologyABSTRACT
Efficient and faithful repair of DNA double-strand breaks (DSBs) is critical for genome stability. To understand whether cells carrying a functional repair apparatus are able to efficiently heal two distant chromosome ends and whether this DNA lesion might result in genome rearrangements, we induced DSBs in genetically modified mouse embryonic stem cells carrying two I-SceI sites in cis separated by a distance of 9 kbp. We show that in this context non-homologous end-joining (NHEJ) can repair using standard DNA pairing of the broken ends, but it also joins 3' non-complementary overhangs that require unusual joining intermediates. The repair efficiency of this lesion appears to be dramatically low and the extent of genome alterations was high in striking contrast with the spectra of repair events reported for two collinear DSBs in other experimental systems. The dramatic decline in accuracy suggests that significant constraints operate in the repair process of these distant DSBs, which may also control the low efficiency of this process. These findings provide important insights into the mechanism of repair by NHEJ and how this process may protect the genome from large rearrangements.
