Damaged DNA-binding Protein DDB Stimulates the Excision of Cyclobutane Pyrimidine Dimers in Vitro in Concert with XPA and Replication Protein A

Abstract

Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, damaged DNA-binding protein (DDB), is a heterodimer of two polypeptides, p127 and p48. Recent in vivo studies suggested that DDB is involved in global genome repair of cyclobutane pyrimidine dimers (CPDs), but the mechanism remains unclear. Here, we show that in vitro DDB directly stimulates the excision of CPDs but not (6 -4)photoproducts. The excision activity of cell-free extracts from Chinese hamster AA8 cell line that lacks DDB activity was increased 3-4-fold by recombinant DDB heterodimer but not p127 subunit alone. Moreover, the addition of XPA or XPA ؉ replication protein A (RPA), which themselves enhanced excision, also enhanced the excision in the presence of DDB. DDB was found to elevate the binding of XPA to damaged DNA and to make a complex with damaged DNA and XPA or XPA ؉ RPA as judged by both electrophoretic mobility shift assays and DNase I protection assays. These results suggest that DDB assists in the recognition of CPDs by core NER factors, possibly through the efficient recruitment of XPA or XPA⅐RPA, and thus stimulates the excision reaction of CPDs. Nucleotide excision repair (NER)1 is the major mechanism for removing bulky DNA lesions including cyclobutane pyrimidine dimers (CPDs) and (6 -4)photoproducts induced by sun light in humans (1-4). The basic reaction mechanism of NER is highly conserved from yeast to human. The core reaction from DNA damage recognition to excision of the damage is accomplished by six repair factors (XPA, RPA, XPC⅐HR23B, TFIIH, XPF⅐ERCC1, XPG), so called excision nuclease (excinuclease) (5-8). The recent development of reconstituted systems using highly purified proteins has enabled analyses of the detailed reaction mechanism of NER (7-10). In yeast, some accessory or regulatory factors such as Rad7⅐Rad16 complex (11-13), ABF1 (14), and MMS19 (15-16) have been reported to help mediate NER. The existence of such factors in humans, however, has not been clearly demonstrated.Damaged DNA-binding protein (DDB) is one of the candidates that might play such an accessory role in human cells. DDB is composed of two subunits, p127 and p48, and has a higher affinity for various types of DNA lesions compared with the damage recognition subunits (XPA, RPA, XPC⅐HR23B, and TFIIH) of the six core repair factors (17-19). DDB activity has been shown to be missing in a subset of xeroderma pigmentosum group E (XP-E) patients. XP is an autosomal recessive disease associated with abnormal sensitivity to UV lights and a high incidence of skin cancer, which is known to be caused by defects in NER. XP-E is one of eight genetic complementation groups (XP-A through XP-G and XP-V) of XP patients, and, along with XP-V, manifests the mildest clinical features and deficiency in NER. XP-E individuals lacking DDB activity were found to carry mutations in p48 gene (20 -23). Because of its high specificity for binding to damaged DNA and a defect in some XP-E cells, DDB has been postulated to be involved...

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

“…In support of this hypothesis, introduction of excess recombinant UV-DDB to in vitro reconstituted NER reactions resulted in inhibition of repair of (6-4) photoproducts (8,9). In addition, in vivo studies of fluorescently tagged UV-DDB binding have reported immobile binding of UV-DDB to DNA for up to 4 h in XP-A cells (41).…”

“…During global genomic repair, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] are repaired by the nucleotide excision repair (NER) pathway that recognizes and excises bulky helix distorting lesions in the genome (6, 7). The recognition of CPD lesions in UV-damaged chromatin is mediated by UV-damaged DNAbinding protein (UV-DDB), composed of the tightly associated heterodimer of damage-specific DNA binding protein (DDB) 1 (p127) and DDB2 (p48) (5,8). Following surveillance and CPD identification by UV-DDB, NER proceeds via lesion handover to XPC-hHR23B-centrin2 (XPC) followed by damage verification, helix opening and stabilizing of the repair intermediates, dual incision of the DNA in the context of the lesion, repair synthesis, and DNA ligation (7).…”

Xeroderma Pigmentosum Complementation Group A Protein (XPA) Modulates RPA-DNA Interactions via Enhanced Complex Stability and Inhibition of Strand Separation Activity

“…However, the degree of relocation and association of XPA with the damaged sites observed in wild type cells was minimal, and therefore the results obtained monitoring XPA relocalization with the XPC cells are difficult to interpret (21). In addition to XPC-hHR23B and the XPA-RPA complex, the damaged DNAbinding protein DDB (heterodimer p127 and p48) also preferentially binds damaged DNA and is specifically involved in the repair of DNA adducts that fail to cause a large change in thermal stability of the duplex DNA (17,41). Whether these protein factors play a role prior to RPA-XPA binding or whether there is a higher order recognition complex remains to be determined.…”

“…There is also evidence that the XPChHR23B protein complex may initiate recognition of DNA damage for the global genomic repair pathway of NER (15). Recent evidence also implicates the DDB heterodimer in damage recognition because the complex binds damaged DNA with high affinity (16) and can dramatically increase the repair rate of certain DNA adducts, including cyclobutane pyrimidine dimers in conjunction with XPA and RPA (17). In addition, the p48 subunit of DDB has been demonstrated to localize to the sites of UV-induced DNA damage independent of XPA and XPC (18).…”

DDB2 gene disruption leads to skin tumors and resistance to apoptosis after exposure to ultraviolet light but not a chemical carcinogen

“…DDB strongly binds to DNA damages caused by UV light, including (6-4) photoproducts and trans,syn-cyclobutane pyrimidine dimers, yet it does not have a strong affinity for cis,syn-cyclobutane pyrimidine dimers, the most abundant UV photoproduct (5,(7)(8)(9)(10)(11).…”