Mechanisms of replication fork protection: a safeguard for genome stability.

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.

Tipin/Tim1/And1 protein complex promotes Pol alpha chromatin binding and sister chromatid cohesion.

The Tipin/Tim1 complex plays an important role in the S-phase checkpoint and replication fork stability. However, the biochemical function of this complex is poorly understood. Using Xenopus laevis egg extract we show that Tipin is required for DNA replication in the presence of limiting amount of replication origins. Under these conditions the DNA replication defect correlates with decreased levels of DNA Polalpha on chromatin. We identified And1, a Polalpha chromatin-loading factor, as new Tipin-binding partner. We found that both Tipin and And1 promote stable binding of Polalpha to chromatin and that this is required for DNA replication under unchallenged conditions. Strikingly, extracts lacking Tipin and And1 also show reduced sister chromatids cohesion. These data indicate that Tipin/Tim1/And1 form a complex that links stabilization of replication fork and establishment of sister chromatid cohesion.

Plx1 is required for chromosomal DNA replication under stressful conditions.

Polo-like kinase (Plk)1 is required for mitosis progression. However, although Plk1 is expressed throughout the cell cycle, its function during S-phase is unknown. Using Xenopus laevis egg extracts, we demonstrate that Plx1, the Xenopus orthologue of Plk1, is required for DNA replication in the presence of stalled replication forks induced by aphidicolin, etoposide or reduced levels of DNA-bound Mcm complexes. Plx1 binds to chromatin and suppresses the ATM/ATR-dependent intra-S-phase checkpoint that inhibits origin firing. This allows Cdc45 loading and derepression of DNA replication initiation. Checkpoint activation increases Plx1 binding to the Mcm complex through its Polo box domain. Plx1 recruitment to chromatin is independent of checkpoint mediators Tipin and Claspin. Instead, ATR-dependent phosphorylation of serine 92 of Mcm2 is required for the recruitment of Plx1 to chromatin and for the recovery of DNA replication under stress. Depletion of Plx1 leads to accumulation of chromosomal breakage that is prevented by the addition of recombinant Plx1. These data suggest that Plx1 promotes genome stability by regulating DNA replication under stressful conditions.

Differences in the DNA replication of unicellular eukaryotes and metazoans: known unknowns.

Although the basic mechanisms of DNA synthesis are conserved across species, there are differences between simple and complex organisms. In contrast to lower eukaryotes, replication origins in complex eukaryotes lack DNA sequence specificity, can be activated in response to stressful conditions and require poorly conserved factors for replication firing. The response to replication fork damage is monitored by conserved proteins, such as the TIPIN-TIM-CLASPIN complex. The absence of this complex induces severe effects on yeast replication, whereas in higher eukaryotes it is only crucial when the availability of replication origins is limiting. Finally, the dependence of DNA replication on homologous recombination proteins such as RAD51 and the MRE11-RAD50-NBS1 complex is also different; they are dispensable for yeast S-phase but essential for accurate DNA replication in metazoans under unchallenged conditions. The reasons for these differences are not yet understood. Here, we focus on some of these known unknowns of DNA replication.

Risk of stillbirth in older mothers: a specific delivery plan might be considered for prevention.

OBJECTIVE: In recent decades, the trend for women is to delay childbearing. However, worldwide, advanced maternal age is an independent risk factor for stillbirth, as well as advanced gestational age. National data are not available about stillbirths in the Italian population. We explored whether, at term of pregnancy, advanced maternal age is associated with an increased risk of stillbirth in Italy. We speculate that a policy of induction of labor at term of pregnancy in older mothers may significantly reduce the stillbirth. METHODS: Data provided by Italian Ministry of Health and National Statistical Institute were used to identify all singleton deliveries ≥22 weeks of gestation during a four years study period. We evaluated the outcome of pregnancy (livebirths or stillbirths) and we stratified data by gestational age and by maternal age at delivery. The hazard risk and the relative risk of stillbirth were calculated. RESULTS: The overall stillbirth rate was 3.4 per 1000, with a total of 6451 cases of stillbirths in the four years study period. Overall, the risk of stillbirth increases at term of pregnancy in all maternal age groups, especially in older mothers. A total of 674 stillbirths occurred in women aged 40 years or older and 24.2% of them (n = 163) occurred at term of pregnancy. Among women aged 40 years and above, 7.3% of stillbirths (49/674) occurred beyond 39 weeks of gestation. The hazard risk doubles from 39 to 40 weeks, from 0.60 per 1000 ongoing pregnancies to 1.16 per 1000 ongoing pregnancies; the relative risk at 40 weeks of gestation was the highest in the older mothers and was 5.17 (95% CI 3.16-8.46). CONCLUSIONS: The effect of maternal age on birth outcomes is a relevant aspect in Italy. If the association between maternal age and stillbirth is supposed to be part of the pathophysiology of fetal death, our data indicate that induction of labor before 40 weeks of gestation in women aged 40 years old or older might prevent overall 7.3% of stillbirths for induction at 39 weeks, 13% of stillbirths for induction at 38 weeks. To reduce potentially preventable stillbirths, caregivers should perform a specific risk assessment for each pregnant woman. The impact of maternal age should be seriously considered, and an individualized approach should be planned at term of pregnancy in older mothers, including the possibility of a slightly anticipation of induction of labor if spontaneously undelivered.

Hereditary spastic paraplegia: clinical genetic study of 15 families.

BACKGROUND: Autosomal dominant hereditary spastic paraplegia (ADHSP) is mainly caused by mutations in the SPG4 gene, which encodes a new member of the AAA (adenosine triphosphatases associated with diverse cellular activities) protein family (spastin). Accumulation of genotype-phenotype correlation is important for better understanding of SPG4-linked hereditary spastic paraplegia. OBJECTIVES: To perform a clinical and genetic study of families with ADHSP and to perform the functional analysis of the founder mutation discovered in the SPG4 gene. DESIGN: Genetic and clinical study. Patients Fifteen unrelated families with ADHSP originating from southern Scotland. MAIN OUTCOME MEASURES: Clinical assessment, linkage analysis, haplotype study, expression of mutant spastin protein in cultured cells. RESULTS: Nine families with ADHSP were linked to the SPG4 locus at 2p21-p24. Sequence analysis of SPG4showed a novel N386S mutation in all 9 of these families. Expression of mutant spastin showed aberrant distribution in cultured cells. Haplotype analysis suggested the existence of a common founder. Clinical examination of the affected members carrying the mutation showed phenotypic variations including broad range of age at onset and disease duration and additional neurologic features such as mental retardation. Magnetic resonance imaging demonstrated unique features, including thin corpus callosum and atrophy of the cerebellum in 2 patients. Linkage and sequence analyses showed no evidence of linkage to the currently known ADHSP loci in the remaining 6 families. CONCLUSIONS: A founder SPG4 mutation N386S was identified in the families with ADHSP originating from southern Scotland. Clinical investigation showed intrafamilial and interfamilial phenotypic variations. The genetic study demonstrated evidence of further genetic heterogeneity in ADHSP.

[The Italian NH Hospital System: efficiency and compliance at a regional level]. / Il sistema ospedaliero del Servizio Sanitario Nazionale: efficienza e appropriatezza a livello regionale.

According to the newly established basic health parameters (Livelli Essenziali di Assistenza, LEA), the authors have assessed efficiency and compliance within the Italian NH hospital system. The first part is focused on the 1999 Demand and Supply, outlining differences between regions, whereas the second part investigates the compliance indicators over the years 1997-99. A general analysis of principal components was also carried out to get a final pattern of the outcomes. Although data only concern a short-term run, the study shows an increase in efficiency and appropriateness within hospital systems, despite existing differences between areas.

Mta2 promotes Tipin-dependent maintenance of replication fork integrity.

Orderly progression of S phase requires the action of replisome-associated Tipin and Tim1 proteins, whose molecular function is poorly understood. Here, we show that Tipin deficiency leads to the accumulation of aberrant replication intermediates known as reversed forks. We identified Mta2, a subunit of the NuRD chromatin remodeler complex, as a novel Tipin binding partner and mediator of its function. Mta2 is required for Tipin-dependent Polymerase α binding to replicating chromatin, and this function is essential to prevent the accumulation of reversed forks. Given the role of the Mta2-NuRD complex in the maintenance of heterochromatin, which is usually associated with hard-to-replicate DNA sequences, we tested the role of Tipin in the replication of such regions. Using a novel assay we developed to monitor replication of specific genomic loci in Xenopus laevis egg extract we demonstrated that Tipin is directly required for efficient replication of vertebrate centromeric DNA. Overall these results suggest that Mta2 and Tipin cooperate to maintain replication fork integrity, especially on regions that are intrinsically difficult to duplicate.