Ulrich Hübscher

Proliferating cell nuclear antigen (PCNA): a dancer with many partners

Proliferating cell nuclear antigen (PCNA) was originally characterised as a DNA sliding clamp for replicative DNA polymerases and as an essential component of the eukaryotic chromosomal DNA replisome. Subsequent studies, however, have revealed its striking ability to interact with multiple partners, which are involved in several metabolic pathways, including Okazaki fragment processing, DNA repair, translesion DNA synthesis, DNA methylation, chromatin remodeling and cell cycle regulation. PCNA in mammalian cells thus appears to play a key role in controlling several reactions through the coordination and organisation of different partners. Two major questions have emerged: how do these proteins access PCNA in a coordinated manner, and how does PCNA temporally and spatially organise their functions? Structural and biochemical studies are starting to provide a first glimpse of how both tasks can be achieved.

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.

Efficient production of chicken egg yolk antibodies against a conserved mammalian protein.

The egg yolk of immunized chicken is a rich and inexpensive source of specific polyclonal antibodies. In this paper we show that 20‐30 μg of a highly conserved mammalian protein, as exemplified by proliferating cell nuclear antigen, are sufficient to induce an immune response. Immunoblot analysis revealed that specific antibodies appeared 20 days after immunization, reached a plateau after 30 days, and remained high until at least day 81. A total amount of 4 g immunoglobulin was extracted from 62 eggs of one immunized hen, yielding approximately 130 mg of specific antibodies.— Gassmann, M.; Thömmes, P.; Weiser, T.; Hübscher, U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 4: 2528‐2532; 1990.

A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage.

ABSTRACT Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control.

Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain‐connecting loop of PCNA and loops on one face of the trimer, close to the C‐termini, is involved in binding to all of the following proteins: DNA polymerase δ, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase δ/PCNA. We demonstrate that PCNA must be located below a 5′ flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C‐termini facing forwards, in the direction of DNA synthesis.

Eukaryotic DNA polymerases, a growing family

In eukaryotic cells, DNA polymerases are required to maintain the integrity of the genome during processes, such as DNA replication, various DNA repair events, translesion DNA synthesis, DNA recombination, and also in regulatory events, such as cell cycle control and DNA damage checkpoint function. In the last two years, the number of known DNA polymerases has increased to at least nine (called alpha, beta, gamma, delta, epsilon, zeta, eta, t and iota), and yeast Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase.

The 3′–5′ exonucleases

Over the past few years, several new 3′–5′ exonucleases have been identified. In vitro studies of these enzymes have uncovered much about their potential functions in vivo, and certain organisms with a defect in 3′–5′ exonucleases have an increased susceptibility to cancer, especially under conditions of stress. Here, we look at not only the newly discovered enzymes, but also at the roles of other 3′–5′ exonucleases in the quality control of DNA synthesis, where they act as proofreading exonucleases for DNA polymerases during DNA replication, repair and recombination.

Mammalian base excision repair by DNA polymerases δ and ε

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase  β (Pol  β )-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol β-deleted mouse fibroblasts to separate subfractions containing either Pol δ or Pol ε. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol δ and Pol ε were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol β. Furthermore, our observations indicated, that either Pol δ and/or Pol ε participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol δ and/or Pol ε are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol β in one-gap filling reactions.

8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols—belonging to the B, Y and X classes—during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols λ and η. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol λ, and 68-fold by DNA pol η, respectively. Experiments with DNA-pol-λ-null cell extracts suggested an important role for DNA pol λ. On the other hand, DNA pol ι, together with DNA pols α, δ and β, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.

The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1–NPL4 and Lys-48-linked ubiquitin (K48–Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48–Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97–UFD1–NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability.