Protein complexes in nucleotide excision repair.

The main pathway by which mammalian cells remove DNA damage caused by UV light and some other mutagens is nucleotide excision repair (NER). The best characterised components of the human NER process are those proteins defective in the inherited disorder xeroderma pigmentosum (XP). The proteins known to be involved in the first steps of the NER reaction (damage recognition and incision-excision) are heterotrimeric RPA, XPA, the 6 to 9 subunit TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF. Many interactions between these proteins have been found in recent years using different methods both in mammalian cells and for the homologous proteins in yeast. There are virtually no quantitative measurements of the relative strengths of these interactions. Higher order associations between these proteins in solution and even the existence of a complete "repairosome" complex have been reported, which would have implications both for the mechanism of repair and for the interplay between NER and other cellular processes. Nevertheless, evidence for a completely pre-assembled functional repairosome in solution is inconclusive and the order of action of repair factors on damaged DNA is uncertain.     

Scanning the DNA for damage by the nucleotide excision repair machinery

Damage detection during nucleotide excision repair requires the action of multiple proteins that probe the DNA for different parameters like disruption of basepairing, DNA bendability and presence of chemical modifications. In a recent study it has been shown that two of these probing events can be spatially separated on the DNA. Upon initial binding of the XPC protein to a region with disrupted basepairing a complex of XPC, TFIIH and XPA is translocated to a CPD lesion even when this chemical modification is located up to 160 nucleotides from the mispaired region.     

Visualization of mismatch repair complexes using fluorescence microscopy

DNA mismatch repair (MMR) is a surveillance mechanism present in most living organisms, which repairs errors introduced by DNA polymerases. Importantly, loss of MMR function due to inactivating mutations and/or epigenetic silencing results in the accumulation of mutations and as consequence increased cancer susceptibility, as observed in Lynch syndrome patients.During the past decades important progress has been made in the MMR field resulting in the identification and characterization of essential MMR components, culminating in the in vitro reconstitution of 5′ and 3′ nick-directed MMR. However, several mechanistic aspects of the MMR reaction remain not fully understood, therefore alternative approaches and further investigations are needed.Recently, the use of imaging techniques and, more specifically, visualization of MMR components in living cells, has broadened our mechanistic understanding of the repair reaction providing more detailed information about the spatio-temporal organization of MMR in vivo. In this review we would like to comment on mechanistic aspects of the MMR reaction in light of these and other recent findings. Moreover, we will discuss the current limitations and provide future perspectives regarding imaging of mismatch repair components in diverse organisms.     

Isolation of human complexes proficient in nucleotide excision repair.

More than 20 polypeptides are required for the process of nucleotide excision repair (NER) in both human and yeast cells. This pathway of excision repair has most often been viewed as an ordered multi-step process involving steps of damage recognition, incision/excision and finally repair DNA synthesis. Here we present evidence for the existence of a complex of human NER proteins pre-assembled in the absence of damaged DNA. This multi-protein complex was initially isolated from HeLa cell extracts by affinity chromatography on a matrix containing the damage recognition protein XPA. Subsequent co-immunoprecipitation and gel filtration experiments demonstrated that a significant portion of the human NER proteins was present in the form of a high molecular weight complex and that these complexes, or repairosomes, were capable of performing all steps of NER in vitro . Consistent with studies indicating that DNA polymerasesdeltaandstraightepsiloncan both function in NER, these two polymerases are found in these repairosome complexes.     

New insights into the spliceosome by single molecule fluorescence microscopy

Splicing is an essential eukaryotic process in which introns are excised from precursors to messenger RNAs and exons ligated together. This reaction is catalyzed by a multi-MegaDalton machine called the spliceosome, composed of 5 small nuclear RNAs (snRNAs) and a core set of ～100 proteins minimally required for activity. Because of the spliceosome's size, its low abundance in cellular extracts, and its highly dynamic assembly pathway, analysis of the kinetics of splicing and the conformational rearrangements occurring during spliceosome assembly and disassembly has proven extraordinarily challenging. Here, we review recent progress in combining chemical biology methodologies with single molecule fluorescence techniques to provide a window into splicing in real time. These methods complement ensemble measurements of splicing in vivo and in vitro to facilitate kinetic dissection of pre-mRNA splicing.     

Automated multidimensional single molecule fluorescence microscopy feature detection and tracking

Characterisation of multi-protein interactions in cellular networks can be achieved by optical microscopy using multidimensional single molecule fluorescence imaging. Proteins of different species, individually labelled with a single fluorophore, can be imaged as isolated spots (features) of different colour light in different channels, and their diffusive behaviour in cells directly measured through time. Challenges in data analysis have, however, thus far hindered its application in biology. A set of methods for the automated analysis of multidimensional single molecule microscopy data from cells is presented, incorporating Bayesian segmentation-based feature detection, image registration and particle tracking. Single molecules of different colours can be simultaneously detected in noisy, high background data with an arbitrary number of channels, acquired simultaneously or time-multiplexed, and then tracked through time. The resulting traces can be further analysed, for example to detect intensity steps, count discrete intensity levels, measure fluorescence resonance energy transfer (FRET) or changes in polarisation. Examples are shown illustrating the use of the algorithms in investigations of the epidermal growth factor receptor (EGFR) signalling network, a key target for cancer therapeutics, and with simulated data.     

ATP-dependent selection between single nucleotide and long patch base excision repair

DNA base excision repair (BER) constitutes a major mechanism to restore the integrity of the genome following modifications of nucleobases. Although it is well established that poly(ADP-ribosylation) facilitates BER, the mechanism of this stimulation has remained unknown. Previous observations suggested that poly(ADP-ribose), which is synthesised from NAD(+), could serve as a unique source of ATP required for the ligation step in BER. This pathway of ATP generation is thought to compensate ATP shortage and relies on the release of pyrophosphate during DNA repair synthesis. Here, we present evidence that, in situations of cellular energy depletion, the synthesis of poly(ADP-ribose) is indeed stimulated. Simultaneously, single nucleotide repair is reduced. Rather, the number of nucleotides incorporated by DNA polymerase beta (Pol beta) during DNA repair synthesis is increased. Using a reconstituted system including the recombinant BER proteins Pol beta, AP endonuclease 1 (APE 1), X-ray repair cross-complementing group-1 (XRCC1), DNA ligase III (Lig III), flap endonuclease 1 (FEN 1), and poly(ADP-ribose) polymerase-1 (PARP-1), it is demonstrated that in the absence of ATP, both long patch DNA synthesis by Pol beta and poly(ADP-ribosylation) catalysed by PARP-1 are stimulated. Consequently, the preferred use of either long patch or single nucleotide BER depends on the availability of ATP. It is proposed that long patch BER is required for ATP generation from poly(ADP-ribose) and, therefore, predominant under conditions of ATP shortage.     

Partially condensed DNA conformations observed by single molecule fluorescence microscopy.

To detect partially condensed conformations of a double-stranded DNA molecule, single molecule fluorescence microscopy is performed here. The single DNA molecules are ethidium stained, 670 kilobase pair bacteriophage G genomes that are observed both during and after expulsion from capsids. Expulsion occurs in an agarose gel. Just after expulsion, the entire G DNA molecule typically has a partially condensed conformation not previously described (called a balloon). A balloon subsequently extrudes a filamentous segment of DNA. The filamentous segment becomes gently elongated via diffusion into the network that forms the agarose gel. The elongated DNA molecule usually has bright spots that undergo both appearance/disappearance and apparent motion. These spots are called dynamic spots. A dynamic spot is assumed to be the image of a zone of partially condensed DNA segments (globule). The positions of globules along an elongated DNA molecule 1) are restricted primarily to time-stable regions with comparatively high thermal motion-induced, micrometer-scale bending of the DNA molecule and 2) move within a given region on a time scale smaller than the time scale of recording. Less mobile globules are observed when either magnesium cation or ethanol is added before gel-embedding DNA molecules. These observations are explained by globules induced at equilibrium by a bending-dependent, inter-DNA segment force. Theory has previously predicted that globules are induced by electrostatic forces along an electrically charged polymer at equilibrium. The hypothesis is proposed that intracellular DNA globules assist action-at-a-distance during DNA metabolism.     

Single long DNA molecule analysis using fluorescence microscopy.

Single long DNA molecule (T4 DNA) in agarose gel was visualized with a fluorescence microscope. We confirmed alternating current electric fields is effective for stretching of single DNA molecule in agarose gel. This stretching phenomenon was observed with wide range of agarose gel concentration from 0.5%(W/V) to 1.5%. From this observation, the presence of agarose gel fiber is essential for this stretching phenomenon. The stretching process of several DNA molecules in gel shows discontinuity, which is never observed in polymer systems. It would be based on topological restriction from gel fibers.     

Single large DNA molecule analysis using fluorescence microscopy.

A large DNA analysis method which enable to obtain spatial information of positions of specific sequences along DNA molecule has been developed. Making use of the phenomenon that large DNA molecule is elongated stably under alternative current field in a concentrated linear polymer solution, direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. Then, the spatial positions of the fluorescence spot of the probe on the DNA molecule were determined by image analysis.     

The eukaryotic nucleotide excision repair pathway

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.     

Chromatin rearrangements during nucleotide excision repair

The removal of DNA damage from the eukaryotic genome requires DNA repair enzymes to operate within the complex environment of chromatin. We review the evidence for chromatin rearrangements during nucleotide excision repair and discuss the extent and possible molecular mechanisms of these rearrangements, focusing on events at the nucleosome level of chromatin structure.     

Ribonucleotides as nucleotide excision repair substrates

The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10–20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2′-OH of the ribonucleotide – with its unique electrostatic and hydrogen bonding properties – may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.     

Unsupervised noise removal algorithms for three-dimensional confocal fluorescence microscopy

Algorithms are presented for effective suppression of the quantum noise artifact that is inherent to three-dimensional confocal fluorescence microscopy images of extended spatial objects such as neurons. The specific advances embodied in these algorithms are as follows: (i) they incorporate an automatic and pattern-constrained three-dimensional segmentation of the image field, and use it to limit any smoothing to the interiors of specified image regions and hence avoid the blurring that is inevitably associated with conventional noise removal algorithms; (ii) they are ‘unsupervised’ in the sense that they automatically estimate and adapt to the unknown spatially and temporally varying noise level in the microscopy data. Fast computation is achieved by parallel computation methods, rather than by algorithmic or modelling compromises.The quantum noise artifact is modelled using a mixture of spatially non-homogeneous Poisson point processes. The intensity of each component process is constrained to lie in specific intervals. A set of segmentation and edge-site variables are used to determine the intensity of the mixture process. Using this model, the noise-removal process is formulated as the joint optimal estimation of the segmentation labels, edge-sites and intensity of the mixture Poisson point process, subject to a combination of stochastic priors and syntactic pattern constraints. The computations proceed iteratively, starting from a set of approximate user-supplied, or default initial guesses of the underlying random process parameters. An Expectation Maximization algorithm is used to obtain a more precise characterization of these parameters, that are then input to a joint estimation algorithm.Stereoscopic renderings of processed three-dimensional images of murine hippocampal neurons are presented to demonstrate the effectiveness of the method. The processed images exhibit increased contrast and significant smoothing and reduction of the background intensity while avoiding any blurring of the foreground neuronal structures.     

Base flipping in nucleotide excision repair

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.     

17 beta-Oestradiol attenuates nucleotide excision repair

Epidemiological studies strongly suggest associations between chronic exposure to endogenous oestrogens and the development of breast and gynaecological tumours. Two mechanisms by which 17 beta-oestradiol (E2) may enhance tumorigenesis are: (i) enhancement of cell proliferation and (ii) the production of reactive, genotoxic metabolites. Here we suggest an additional mechanism, inhibition of DNA repair. The removal of UV-induced thymine dimers from human keratinocytes, reflective of nucleotide excision repair, was significantly attenuated by treatment of cells with E2. In contrast, treatment with 17 alpha-oestradiol had no effect. Mechanisms are proposed for this effect of E2, which may contribute to its carcinogenic potential.