Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase h (polh), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase i (poli), like polh, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that polh and poli foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poli in replication foci is largely dependent on the presence of polh. Using several different approaches, we demonstrate that polh and poli interact with each other physically and that the C-terminal 224 amino acids of poli are suf®cient for both the interaction with polh and accumulation in replication foci. Our results provide strong evidence that polh targets poli to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis. Keywords: DNA polymerase/replication foci/UV light/ xeroderma pigmentosum variants Introduction DNA damage occurs ubiquitously in all cells. In order to maintain the stability of the genome, cells have evolved mechanisms not only to repair all types of DNA damage, but also to replicate DNA from which the damage has not been removed (post-replication repair). In the case of human cells, a major mechanism for carrying out postreplication repair involves translesion synthesis (TLS) past damaged sites. TLS is de®cient in the variant form of the sun-sensitive cancer-prone disorder xeroderma pigmentosum (XP). The gene defective in these XP variants (XP-V) encodes a DNA polymerase, polh (Johnson et al., 1999;Masutani et al., 1999), which is able to replicate undamaged templates or those containing cyclobutane pyrimidine dimers (CPDs, the major UV photoproduct) with equal ef®ciencies (Masutani et al., 1999). TLS by polh is the principal mechanism for bypassing CPDs in human cells. Although the lack of polh in XP-V cells does not confer substantial hypersensitivity to killing by UV light, UV hypermutability is increased to levels approaching those in classical XP cells, which are de®cient in nucleotide excision repair (Maher et al., 1976).Polh is a member of the recently discovered Y-family of DNA polymerases (Ohmori et al., 2001), which have been best characterized for their lesion-bypassing properties (reviewed in Goodman, 2002). There are, however, few studies to date to indicate how these polymerases function inside cells. In previous work, we showed that in S-phase cells, polh localizes in replication foci. On exposure to DNA-damaging treatments, we observed an accumulation of polh-containing foci. These appear to represent replication factories in which replication forks are stalled at lesions (Kannouche et al., 2001). The C-terminal 70 amino acids of polh are requ...

SummaryThe zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186, in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macro- phages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.

Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion. The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference. Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second. Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.