Two-step ATP-driven opening of cohesin head.

The cohesin ring is a protein complex composed of four core subunits: Smc1A, Smc3, Rad21 and Stag1/2. It is involved in chromosome segregation, DNA repair, chromatin organization and transcription regulation. Opening of the ring occurs at the "head" structure, formed of the ATPase domains of Smc1A and Smc3 and Rad21. We investigate the mechanisms of the cohesin ring opening using techniques of free molecular dynamics (MD), steered MD and quantum mechanics/molecular mechanics MD (QM/MM MD). The study allows the thorough analysis of the opening events at the atomic scale: i) ATP hydrolysis at the Smc1A site, evaluating the role of the carboxy-terminal domain of Rad21 in the process; ii) the activation of the Smc3 site potentially mediated by the movement of specific amino acids; and iii) opening of the head domains after the two ATP hydrolysis events. Our study suggests that the cohesin ring opening is triggered by a sequential activation of the ATP sites in which ATP hydrolysis at the Smc1A site induces ATPase activity at the Smc3 site. Our analysis also provides an explanation for the effect of pathogenic variants related to cohesinopathies and cancer.

Identification and Functional Characterization of Two Intronic NIPBL Mutations in Two Patients with Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronic NIPBL mutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13)_(-8) deletion. While the loss of exon 28 retains the reading frame of the NIBPL transcript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the total NIPBL mRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.

New case of mitochondrial HMG-CoA synthase deficiency. Functional analysis of eight mutations.

Mitochondrial HMG-CoA synthase deficiency is a rare inherited metabolic disorder that affects ketone-body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycaemia, dicarboxylic aciduria, and in severe cases, coma. This deficiency may have been under-diagnosed owing to the absence of specific clinical and biochemical markers, limitations in liver biopsy and the lack of an effective method of expression and enzyme assay for verifying the mutations found. To date, eight patients have been reported with nine allelic variants of the HMGCS2 gene. We present a new method of enzyme expression and a modification of the activity assay that allows, for first time, the functional study of missense mutations found in patients with this deficiency. Four of the missense mutations (p.V54M, p.R188H, p.G212R and p.G388R) did not produce proteins that could have been detected in soluble form by western blot; three produced a total loss of activity (p.Y167C, p.M307T and p.R500H) and one, variant p.F174L, gave an enzyme with a catalytic efficiency of 11.5%. This indicates that the deficiency may occur with partial loss of activity of enzyme. In addition, we describe a new patient with this deficiency, in which we detected the missense allelic variant, c.1162G>A (p.G388R) and the nonsense variant c.1270C>T (p.R424X).