Vladimir N. Podust

Mammalian DNA nucleotide excision repair reconstituted with purified protein components

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Nedd8 Modification of Cul-1 Activates SCFβTrCP-Dependent Ubiquitination of IκBα

ABSTRACT Regulation of NF-κB occurs through phosphorylation-dependent ubiquitination of IκBα, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IκBα is carried out by a ubiquitin-ligase enzyme complex called SCFβTrCP . Here we show that Nedd8 modification of the Cul-1 component of SCFβTrCP is important for function of SCFβTrCP in ubiquitination of IκBα. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCFβTrCP , phosphorylated IκBα and β-catenin, indicating that Nedd8–Cul-1 conjugates are part of SCFβTrCP in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed βTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IκBα required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCFβTrCP containing a K720R mutant of Cul-1 only weakly supported IκBα ubiquitination compared to SCFβTrCP containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCFβTrCP . These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IκBα.

DNA unwinding activity of replication protein A

Replication protein A (RP‐A) is a heterotrimeric complex conserved in cukaryotic cells. It binds to single‐stranded DNA and is essential for initiation and elongation of DNA replication. In this communication we give evidence that this protein can unwind DNA independent of magnesium and ATP, two essential cofactors for bona fide DNA helicase activity. RP‐A can unwind up to at least 350 basepairs and appears to be required in stoichiometric amounts. The reaction is extremely sensitive to NaCl and MgCl2. This activity of RF‐A is suggestive for a possible unwinding function in initiation of DNA replication in eukaryotes.

Role of the p68 Subunit of Human DNA Polymerase α-Primase in Simian Virus 40 DNA Replication

ABSTRACT DNA polymerase α-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.

17 DNA Replication Accessory Proteins

INTRODUCTION DNA replication requires the concerted action of many enzymes, as well as other protein and non-protein cofactors. The DNA, in preparation for DNA synthesis, has to become single-strand to serve as a template for the replicative DNA polymerases (pols). It is this form of the DNA that is especially prone to damage of any kind. Nature has provided a set of proteins that support the replicative pols in performing processive, accurate, and rapid DNA synthesis. Furthermore, such proteins also prevent damage to the transient single-strand (ss) DNA. These proteins are called DNA replication accessory proteins. The three best known are the proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and replication protein A (RP-A). In this chapter, we focus on these three protein classes and compare them to their selected counterparts in eukaryotic viruses. Additional replication proteins that also assist the proper function of pols, such as the 3′ → 5′ exonuclease, DNA primase, RNase H, 5′ → 3′ exonuclease, DNA helicases, DNA ligases, and DNA topoisomerases, are covered in various other chapters. Early Discovery of Replication Accessory Proteins in Prokaryotes by Genetics and Defined In Vitro Replication Systems Fifteen years ago it was realized that bacteriophages of Escherichia coli provide a window to understand the cellular events of DNA replication (Kornberg and Baker 1992). By using ssDNA from φX174, G4, and M13 as model replicons, the requirements for a ssDNA-binding protein (SSB) and a DNA synthesis complex were identified. The latter includes the pol III holoenzyme, which...

DNA polymerase δ and ɛ holoenzymes from calf thymus

Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) δ completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (orE.coli singlestrand DNA binding protein, SSB). Pol ɛ core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol ɛ was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol ɛ by PCNA/RF-C/SSB appears to be the superposition of two events: pol, ɛ holoenzyme (pol ɛ, PCNA, RF-C) synthesized longer products than its pol ɛ core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol α/primase with pol δ or pol ɛ holoenzymes on unprimed M13 DNA. While pol δ displayed higher dNMP incorporation than pol ɛ, when a single primer was preannealed to DNA, pol ɛ was more efficient in the utilization of the primers synthesized by pol α/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol ɛ holoenzyme, than for pol δ. Our data support the hypothesis of pol δ as the leading and pol ɛ as the second lagging strand replication enzyme.