DNA molecular combing-based replication fork directionality profiling.

The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

FANCD2 binding identifies conserved fragile sites at large transcribed genes in avian cells.

Common Chromosomal Fragile Sites (CFSs) are specific genomic regions prone to form breaks on metaphase chromosomes in response to replication stress. Moreover, CFSs are mutational hotspots in cancer genomes, showing that the mutational mechanisms that operate at CFSs are highly active in cancer cells. Orthologs of human CFSs are found in a number of other mammals, but the extent of CFS conservation beyond the mammalian lineage is unclear. Characterization of CFSs from distantly related organisms can provide new insight into the biology underlying CFSs. Here, we have mapped CFSs in an avian cell line. We find that, overall the most significant CFSs coincide with extremely large conserved genes, from which very long transcripts are produced. However, no significant correlation between any sequence characteristics and CFSs is found. Moreover, we identified putative early replicating fragile sites (ERFSs), which is a distinct class of fragile sites and we developed a fluctuation analysis revealing high mutation rates at the CFS gene PARK2, with deletions as the most prevalent mutation. Finally, we show that avian homologs of the human CFS genes despite their fragility have resisted the general intron size reduction observed in birds suggesting that CFSs have a conserved biological function.

Hsm3/S5b participates in the assembly pathway of the 19S regulatory particle of the proteasome.

The 26S proteasome, the central enzyme of the ubiquitin-proteasome system, is comprised of the 20S catalytic core particle (CP) and the 19S regulatory particle (RP), itself composed of two subcomplexes, the base and the lid. 20S proteasome assembly is assisted by several chaperones. Integral subunits of the RP participate in its assembly, but no external factors have been identified so far. Here we characterize the yeast Hsm3 protein, which displays unique features regarding 19S assembly. Hsm3 associates with 19S subcomplexes via a carboxy-terminal domain of the Rpt1 base subunit but is missing in the final 26S proteasome. Moreover, Hsm3 is specifically required for the base subcomplex assembly. Finally, we identify the putative species-specific 19S subunit S5b as a functional homolog of the Hsm3 chaperone in mammals. These findings shed light on chaperone-assisted proteasome assembly in eukaryotes.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells.

Common fragile sites: mechanisms of instability revisited.

Common fragile sites (CFSs) are large chromosomal regions prone to breakage upon replication stress that are considered a driving force of oncogenesis. CFSs were long believed to contain sequences blocking fork progression, thus impeding replication completion and leading to DNA breaks upon chromosome condensation. However, recent studies show that delayed completion of DNA replication instead depends on a regional paucity in initiation events. Because the distribution and the timing of these events are cell type dependent, different chromosomal regions can be committed to fragility in different cell types. These new data reveal the epigenetic nature of CFSs and open the way to a reevaluation of the role played by these sites in the formation of chromosome rearrangements found in tumors from different tissues.

Updating the mechanisms of common fragile site instability: how to reconcile the different views?

Common fragile sites (CFSs) are large chromosomal regions long identified by conventional cytogenetics as sequences prone to breakage in cells subjected to replication stress. The interest in CFSs came from their key role in the formation of DNA damage, resulting in chromosomal rearrangements. The instability of CFSs was notably correlated with the appearance of genome instability in precancerous lesions and during tumor progression. Identification of the molecular mechanisms responsible for their instability therefore represents a major challenge. A number of data show that breaks result from mitotic entry before replication completion but the mechanisms responsible for such delayed replication of CFSs and relaxed checkpoint surveillance are still debated. In addition, clues to the molecular events leading to breakage just start to emerge. We present here the results of recent reports addressing these questions.

Dual functions of the Hsm3 protein in chaperoning and scaffolding regulatory particle subunits during the proteasome assembly.

The 26S proteasome, a molecular machine responsible for regulated protein degradation, consists of a proteolytic core particle (20S CP) associated with 19S regulatory particles (19S RPs) subdivided into base and lid subcomplexes. The assembly of 19S RP base subcomplex is mediated by multiple dedicated chaperones. Among these, Hsm3 is important for normal growth and directly targets the carboxyl-terminal (C-terminal) domain of Rpt1 of the Rpt1-Rpt2-Rpn1 assembly intermediate. Here, we report crystal structures of the yeast Hsm3 chaperone free and bound to the C-terminal domain of Rpt1. Unexpectedly, the structure of the complex suggests that within the Hsm3-Rpt1-Rpt2 module, Hsm3 also contacts Rpt2. We show that in both yeast and mammals, Hsm3 actually directly binds the AAA domain of Rpt2. The Hsm3 C-terminal region involved in this interaction is required in vivo for base assembly, although it is dispensable for binding Rpt1. Although Rpt1 and Rpt2 exhibit weak affinity for each other, Hsm3 unexpectedly acts as an essential matchmaker for the Rpt1-Rpt2-Rpn1 assembly by bridging both Rpt1 and Rpt2. In addition, we provide structural and biochemical evidence on how Hsm3/S5b may regulate the 19S RP association to the 20S CP proteasome. Our data point out the diverse functions of assembly chaperones.

Genome-wide mapping of individual replication fork velocities using nanopore sequencing.

Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed, a nanopore sequencing-based method to map and extract the velocity of individual forks detected as tracks of the thymidine analogue bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae mean fork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficient bromodeoxyuridine incorporation and wild-type growth, and precisely quantifies speed changes in cells with altered replisome progression or exposed to hydroxyurea. The positioning of >125,000 fork velocities provides a genome-wide map of fork progression based on individual fork rates, showing a uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites.

Transcription-dependent regulation of replication dynamics modulates genome stability.

Common fragile sites (CFSs) are loci that are hypersensitive to replication stress and hotspots for chromosomal rearrangements in cancers. CFSs replicate late in S phase, are cell-type specific and nest in large genes. The relative impact of transcription-replication conflicts versus a low density in initiation events on fragility is currently debated. Here we addressed the relationships between transcription, replication, and instability by manipulating the transcription of endogenous large genes in chicken and human cells. We found that inducing low transcription with a weak promoter destabilized large genes, whereas stimulating their transcription with strong promoters alleviated instability. Notably, strong promoters triggered a switch to an earlier replication timing, supporting a model in which high transcription levels give cells more time to complete replication before mitosis. Transcription could therefore contribute to maintaining genome integrity, challenging the dominant view that it is exclusively a threat.

Common fragile site profiling in epithelial and erythroid cells reveals that most recurrent cancer deletions lie in fragile sites hosting large genes.

Cancer genomes exhibit numerous deletions, some of which inactivate tumor suppressor genes and/or correspond to unstable genomic regions, notably common fragile sites (CFSs). However, 70%-80% of recurrent deletions cataloged in tumors remain unexplained. Recent findings that CFS setting is cell-type dependent prompted us to reevaluate the contribution of CFS to cancer deletions. By combining extensive CFS molecular mapping and a comprehensive analysis of CFS features, we show that the pool of CFSs for all human cell types consists of chromosome regions with genes over 300 kb long, and different subsets of these loci are committed to fragility in different cell types. Interestingly, we find that transcription of large genes does not dictate CFS fragility. We further demonstrate that, like CFSs, cancer deletions are significantly enriched in genes over 300 kb long. We now provide evidence that over 50% of recurrent cancer deletions originate from CFSs associated with large genes.

Replication dynamics: biases and robustness of DNA fiber analysis.

The factors that govern replication programs are still poorly identified in metazoans, especially in mammalian cells. Thanks to molecular combing, the dynamics of DNA replication can be assessed at the genome-scale level from the cumulative analysis of single DNA fibers. This technique notably enables measurement of replication fork speed and fork asymmetry and that of distances separating either initiation or termination events. The results presented here aim to evaluate requirements critical to accurate measurement of replication parameters by molecular combing. We show that sample size, fiber length and DNA counterstaining are crucial to gain robust information concerning replication dynamics. Our results thus provide a methodological frame to investigate the DNA replication program through molecular combing analyses.

20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals.

The 20S proteasome is the catalytic core of the 26S proteasome, a central enzyme in the ubiquitin-proteasome system. Its assembly proceeds in a multistep and orderly fashion. Ump1 is the only well-described chaperone dedicated to the assembly of the 20S proteasome in yeast. Here, we report a phenotype related to the DNA damage response that allowed us to isolate four other chaperones of yeast 20S proteasomes, which we named Poc1-Poc4. Poc1/2 and Poc3/4 form two pairs working at different stages in early 20S proteasome assembly. We identify PAC1, PAC2, the recently described PAC3, and an uncharacterized protein that we named PAC4 as functional mammalian homologs of yeast Poc factors. Hence, in yeast as in mammals, proteasome assembly is orchestrated by two pairs of chaperones acting upstream of the half-proteasome maturase Ump1. Our findings provide evidence for a remarkable conservation of a pairwise chaperone-assisted proteasome assembly throughout evolution.