In-solution analysis of conformational changes of NRPS adenylation and peptidyl-carrier protein domains under catalytic conditions reveals a new intermediary conformation.
Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent inSAMHD1−/−mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.
Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly-line biosynthesis of a plethora of bioactive peptides. It is poorly understood how catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX-MS analysis, we discovered dynamic mixtures of conformations that undergo distinct population changes in favor of the PCP-A and PCP-C interactions upon completion of the adenylation and thiolation reactions, respectively. To probe this model we blocked PCP binding to the C domain by photocaging and triggered peptide bond formation with light. Changing intramodular domain affinities of the PCP appear to result in conformational shifts according to the logic of the templated assembly process.
The thiol group of the cysteine side chain is arguably the most versatile chemical handle in proteins. To expand the scope of established and commercially available thiol bioconjugation reagents, we genetically encoded a second such functional moiety in form of a latent thiol group that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated and its latent thiol group unmasked on purified proteins using penicillin G acylase (PGA). The enzymatic deprotection depends on steric accessibility, but can occur efficiently within minutes on exposed positions in flexible sequences. The freshly liberated thiol group does not require treatment with reducing agents. We demonstrate the potential of this approach for protein modification with conceptually new schemes for regioselective dual labeling, thiol bioconjugation in presence of a preserved disulfide bond and formation of a novel intramolecular thioether crosslink.
MtDNA integrity is important for mitochondrial function. We analysed mtDNA over the course of mouse lifespan and found that its integrity, as measured by electrophoretic mobility under denaturing conditions, decreased significantly in old animals whereas its ribonucleotide (rNMP) content increased with age. To investigate whether the rNMPs incorporated into mtDNA were responsible for this loss of integrity in old animals, we used mice deficient in SAMHD1, a dNTPase that decreases rNTP/dNTP ratios. Knockout of SAMHD1 almost completely eliminated rNMPs from mtDNA, but did not restore mtDNA integrity in older animals, indicating that rNMP incorporation does not explain the loss of mtDNA integrity. Neither did mtDNA from older animals contain more gaps, deletions or double-strand breaks than that of younger animals. By contrast, mtDNA integrity was restored by treatment with ligase. Thus, we conclude that the loss of mtDNA integrity in old mice is caused by accumulation of nicks. The genome of mitochondria in mammalian cells is a circular 16 kb dsDNA molecule present in multiple copies per cell. This mitochondrial (mt)DNA encodes key subunits of the respiratory chain complexes that synthesize the majority of the cell's ATP. Consequently, a partial loss of the number of copies of mtDNA from the cell (i.e. depletion) or defects in the quality of the DNA can affect energy production and manifest as human disease [1]. MtDNA quality also decreases with age due to the progressive accumulation of deletions and point mutations [2][3][4], which may contribute to the physiology of aging [5].Ribonucleotides (rNMPs) incorporated into DNA are an important threat to genome stability. Although replicative DNA polymerases are highly selective for dNTPs, the large excess of rNTPs over dNTPs in the cell results in incorporation of some rNMPs during DNA replication [6,7]. Due to their reactive 2ʹ-hydroxyl group, these rNMPs increase the risk of strand breaks by several orders of magnitude [8]. Furthermore, the presence of rNMPs in DNA can alter its local structure and elasticity [9][10][11], thus interfering with processes such as replication, transcription or DNA repair. To prevent these negative effects of incorporated rNMPs on genome stability, they are removed from nuclear DNA (nDNA) by the ribonucleotide excision repair pathway, which is initiated by cleavage at the incorporated rNMP by RNase H2 [7,12]. RNase H2 is essential for genome stability in mammals and its absence results in increased single-stranded DNA (ssDNA) breaks and an activated DNA damage response [13,14].In contrast to nDNA, mtDNA contains persistent rNMPs that are incorporated during replication by the mtDNA polymerase, Pol ɣ (reviewed in [15]). Human Pol ɣ is highly selective against using rNTPs as substrates and thus incorporates fewer rNMPs than nuclear replicative DNA polymerases do [16][17][18]. Once incorporated, however, rNMPs persist in mtDNA because mitochondria lack repair pathways for their removal [19,20]. Although a certain level of rNMPs is tolerate...
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