Aziz Sancar

Nucleotide excision repair.

Publisher Summary DNA damage is a common occurrence that compromises the functional integrity of DNA. It is not surprising then that cells have multiple mechanisms for coping with DNA damage, including those introduced by sunlight and other environmental agents. Nucleotide excision repair is a pluripotent pathway for the recognition and removal of a broad spectrum of DNA lesions. Nucleotide excision repair involves the removal of damaged DNA bases by dual incisions bracketing the damaged base, release of the damaged base in the form of 12–13 nucleotide-long oligomers in prokaryotes and 24– 32 nucleotide-long oligomers in eukaryotes followed by polymerase-mediated replacement of the excised nucleotides and sealing the repair patch with ligase. Structural work currently being carried out by numerous groups is expected to provide an understanding at the atomic level for both prokaryotic and eukaryotic excision nucleases, which will aid in designing further biochemical experiments to refine current models.

Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.

Coupling of Human Circadian and Cell Cycles by the Timeless Protein

ABSTRACT The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. We have found that the human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein.

A role for cryptochromes in sleep regulation

BackgroundThe cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2-/-) lack circadian rhythms. We studied sleep in cry1,2-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes.ResultsUnder all three conditions, cry1,2-/- mice exhibit the hallmarks of high non-REM sleep (NREMS) drive (i.e., increases in NREMS time, NREMS consolidation, and EEG delta power during NREMS). This unexpected phenotype was associated with elevated brain mRNA levels of period 1 and 2 (per1,2), and albumin d-binding protein (dbp), which are known to be transcriptionally inhibited by CRY1,2. To further examine the relationship between circadian genes and sleep homeostasis, we examined wild type mice and rats following sleep deprivation and found increased levels of per1,2 mRNA and decreased levels of dbp mRNA specifically in the cerebral cortex; these changes subsided with recovery sleep. The expression of per3, cry1,2, clock, npas2, bmal1, and casein-kinase-1ε did not change with sleep deprivation.ConclusionsThese results indicate that mice lacking cryptochromes are not simply a genetic model of circadian arrhythmicity in rodents and functionally implicate cryptochromes in the homeostatic regulation of sleep.

Human Transcription-Repair Coupling Factor CSB/ERCC6 Is a DNA-stimulated ATPase but Is Not a Helicase and Does Not Disrupt the Ternary Transcription Complex of Stalled RNA Polymerase II

Transcription is coupled to repair in Escherichia coli and in humans. Proteins encoded by the mfd gene in E. coli and by the ERCC6/CSB gene in humans, both of which possess the so-called helicase motifs, are required for the coupling reaction. It has been shown that the Mfd protein is an ATPase but not a helicase and accomplishes coupling, in part, by disrupting the ternary complex of E. coli RNA polymerase stalled at the site of DNA damage. In this study we overproduced the human CSB protein using the baculovirus vector and purified and characterized the recombinant protein. CSB has an ATPase activity that is stimulated strongly by DNA; however, it neither acts as a helicase nor does it dissociate stalled RNA polymerase II, suggesting a coupling mechanism in humans different from that in prokaryotes. CSB is a DNA-binding protein, and it also binds to XPA, TFIIH, and the p34 subunit of TFIIE. These interactions are likely to play a role in recruiting repair proteins to ternary complexes formed at damage sites.

Purification and characterization of human DNA damage checkpoint Rad complexes

Checkpoint Rad proteins function early in the DNA damage checkpoint signaling cascade to arrest cell cycle progression in response to DNA damage. This checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the damage sensor function of the human checkpoint Rad proteins, we purified a heteropentameric complex composed of hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric complex composed of hRad9-hHus1-hRad1 (checkpoint 9-1-1 complex). hRad17-RFC binds to DNA, with a preference for primed DNA and possesses weak ATPase activity that is stimulated by primed DNA and single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1 heterotrimer reminiscent of the replication factor C/proliferating cell nuclear antigen clamp loader/sliding clamp complex of the replication machinery. These findings constitute biochemical support for models regarding the roles of checkpoint Rads as damage sensors in the DNA damage checkpoint response of human cells.

A cryptochrome/photolyase class of enzymes with single-stranded DNA-specific photolyase activity

Photolyases and cryptochrome blue-light photoreceptors are evolutionarily related flavoproteins that perform distinct functions. Photolyases repair UV-damaged DNA in many species from bacteria to plants and animals. Cryptochromes regulate growth and development in plants and the circadian clock in animals. Recently, a new branch of the photolyase/cryptochrome family was identified. Members of this branch exhibited no or trace levels of DNA repair activity in vivo and in vitro and, therefore, were considered to be cryptochromes, and they were named cryptochrome-DASH. Here, we show that Cry-DASH proteins from bacterial, plant, and animal sources actually are photolyases with high degree of specificity for cyclobutane pyrimidine dimers in ssDNA.

The Human Tim/Tipin Complex Coordinates an Intra-S Checkpoint Response to UV That Slows Replication Fork Displacement

ABSTRACT UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

Order of assembly of human DNA repair excision nuclease.

Human excision nuclease removes DNA damage by concerted dual incisions bracketing the lesion. The dual incisions are accomplished by sequential and partly overlapping actions of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF·ERCC1. Of these, RPA, XPA, and XPC have specific binding affinity for damaged DNA. To learn about the role of these three proteins in damage recognition and the order of assembly of the excision nuclease, we measured the binding affinities of XPA, RPA, and XPC to a DNA fragment containing a single (6-4) photoproduct and determined the rate of damage excision under a variety of reaction conditions. We found that XPC has the highest affinity to DNA and that RPA has the highest selectivity for damaged DNA. Under experimental conditions conducive to binding of either XPA + RPA or XPC to damaged DNA, the rate of damage removal was about 5-fold faster for reactions in which XPA + RPA was the first damage recognition factor presented to DNA compared with reactions in which XPC was the first protein that had the opportunity to bind to DNA. We conclude that RPA and XPA are the initial damage sensing factors of human excision nuclease.

Characterization of Reaction Intermediates of Human Excision Repair Nuclease

Nucleotide excision repair in humans is a complex reaction involving 14 polypeptides in six repair factors for dual incisions on either sides of a DNA lesion. To identify the reaction intermediates that form by the human excision repair nuclease, we adopted three approaches: purification of functional DNA·protein complexes, permanganate footprinting, and the employment as substrate of presumptive DNA reaction intermediates containing unwound sequences 5′ to, 3′ to, or encompassing the DNA lesion. The first detectable reaction intermediate was formed by substrate binding of XPA, RPA, XPC·HHR23B plus TFIIH (preincision complex 1, PIC1). In this complex the DNA was unwound on either side of the lesion by no more than 10 bases. Independent of the XPG nuclease function, the XPG protein stabilized this complex, forming a long lived preincision complex 2 (PIC2). The XPF·ERCC1 complex bound to PIC2, forming PIC3, which led to dual incisions and the release of the excised oligomer. With partially unwound DNAs, thymine cyclobutane dimer was excised at a fast rate independent of XPC·HHR23B, indicating that a major function of this protein is to stabilize the unwound DNA or to aid lesion unwinding in preincision complexes.