PAR proteins diffuse freely across the anterior–posterior boundary in polarized C. elegans embryos

FRAP reveals that a stable PAR boundary requires balancing diffusive flux of PAR proteins between domains with spatial differences in PAR protein membrane affinities

Translesion synthesis in mammalian cells

DNA damage blocks the progression of the replication fork. In order to circumvent the damaged bases, cells employ specialized low stringency DNA polymerases, which are able to carry out translesion synthesis (TLS) past different types of damage. The five polymerases used in TLS in human cells have different substrate specificities, enabling them to deal with many different types of damaged bases. PCNA plays a central role in recruiting the TLS polymerases and effecting the polymerase switch from replicative to TLS polymerase. When the fork is blocked PCNA gets ubiquitinated. This increases its affinity for the TLS polymerases, which all have novel ubiquitin-binding motifs, thereby facilitating their engagement at the stalled fork to effect TLS

ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage–induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage–induced checkpoint activation and translesion synthesis in mammalian cells

The role of Schizosaccharomyces pombe SUMO ligases in genome stability

SUMOylation is a post-translational modification that affects a large number of proteins, many of which are nuclear. While the role of SUMOylation is beginning to be elucidated, it is clear that understanding the mechanisms that regulate the process is likely to be important. Control of the levels of SUMOylation is brought about through a balance of conjugating and deconjugating activities, i.e. of SUMO (small ubiquitin-related modifier) conjugators and ligases versus SUMO proteases. Although conjugation of SUMO to proteins can occur in the absence of a SUMO ligase, it is apparent that SUMO ligases facilitate the SUMOylation of specific subsets of proteins. Two SUMO ligases in Schizosaccharomyces pombe, Pli1 and Nse2, have been identified, both of which have roles in genome stability. We report here on a comparison between the properties of the two proteins and discuss potential roles for the proteins

Regulation of Translesion Synthesis DNA Polymerase η by Monoubiquitination

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin

DNA repair, DNA replication and human disorders: A personal journey

I was born in 1946 and grew up in the industrial north-west of England close to the city of Manchester. My parents were German- Jewish refugees, who left Germany fairly early, in 1933. My father helped to establish and was one of the directors of a tannery, which made leather for shoes and handbags. This was part of a group of tanneries established first in Strasbourg by my great-grandfather Ferdinand Oppenheimer. I would describe my childhood and adolescent years as comfortable by general post-war standards. I went to a state primary school and obtained a scholarship to Manchester Grammar School (MGS), a fairly prestigious secondary school. As a child I was always interested in chemistry but had little interest in or knowledge of biology. The educational system in the UK at that time was such that one had to specialise very early and as a consequence I have had no formal biology education since the age of 12, something I have managed to hide reasonably successfully for the rest of my life! In my final two years at MGS I studied just physics, chemistry and mathematics and obtained a scholarship to Pembroke College, Cambridge (England) to study Natural Sciences, with the intention of becoming a chemist. In the second year at Cambridge, one of the options was a course on biochemistry. Having no real idea what this was, I read a book about it in the summer of 1965, and was truly astonished and excited to discover that the basis of life was just a bunch of rather complicated organic chemistry reactions. So I took the biochemistry course in my second year. By the end of that year, I was fed up with chemistry and for my final year I chose to do biochemistry rather than chemistry, a decision I have not regretted. The biochemistry lectures must have been pretty up-to-date, as we were told briefly about the discovery of DNA repair by Dick Setlow [1], a topic that seemed rather esoteric at the time

SUMO chain formation is required for response to replication arrest in S. pombe

SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved betabetaalphabetabetaalphabeta fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pomb

HDM2 ERKs PCNA

In this issue, a study by Groehler and Lannigan (2010. J. Cell Biol. doi:10.1083/jcb.201002124) sheds light on the regulation of proliferating cell nuclear antigen (PCNA) turnover and how it is counteracted by the small chromatin-bound kinase ERK8 (extracellular signal-regulated kinase 8). Importantly, inactivation of ERK8 results in genome instability and is associated with cell transformation

Requirement of Replication Checkpoint Protein Kinases Mec1/Rad53 for Postreplication Repair in Yeast

DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that postreplication repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint