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 XPA.RPA, and thus stimulates the excision reaction of CPDs.
Highlights
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 [1,2,3,4]
Damaged DNA-binding protein (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, replication protein A (RPA), XPC1⁄7HR23B, and TFIIH) of the six core repair factors [17,18,19]
Recent in vivo studies clearly showed that xeroderma pigmentosum group E (XP-E) cells and rodent cells lacking DDB activity are selectively defective in global genome repair (GGR) of CPDs, and transfection of the p48 gene into the rodent cells complements the deficiencies [28, 29], suggesting that DDB may be involved in GGR especially for CPDs
Summary
Plasmid Constructs—To overproduce FLAG-tagged DDB subunits in insect cells, 5Ј-terminal portions of cDNAs with FLAG epitope se-. B, 32P-labeled 56-bp duplex DNA (2 fmol) either with or without a (6 – 4)photoproduct was incubated at 30 °C for 20 min with 10 g of CFE prepared from HeLa (lanes 3 and 4) or AA8 (lanes 5 and 6) cell lines or 70 ng of recombinant DDB heterodimer (lanes 7 and 8) and p127 subunit (lanes 9 and 10). The standard reaction mixture contained 3– 6 fmol of 136-bp substrate, the indicated recombinant proteins, and 50 g of CFE prepared from AA8 cells in 25 l of reaction buffer (32 mM Hepes-KOH (pH 7.9), 64 mM KCl, 6.44 mM MgCl2, 0.16 mM dithiothreitol, 0.16 mM EDTA, 2 mM ATP, and 4% glycerol). Electrophoretic Mobility Shift Assay—Two fmol of 32P-labeled 56-bp substrates were incubated with the indicated amounts of recombinant proteins or CFE at 30 °C for 20 min in 25 l of reaction buffer. The products were purified, separated on 10% denaturing polyacrylamide gels, and analyzed by autoradiography
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
