TAC102 is a novel component of the mitochondrial genome segregation machinery in trypanosomes

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization

Is Thymidine Glycol Containing DNA a Substrate of <i>E. coli</i> DNA Mismatch Repair System?

<div><p>The DNA <u>m</u>is<u>m</u>atch <u>r</u>epair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for <i>E. coli</i> MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the <i>E.</i> coli MMR system. Probably, the MutS transformation to active “sliding clamp” conformation on Tg-DNA is problematic.</p></div

Fluorescence emission spectra.

<p>Panels <b>A–F</b> correspond to DNA duplexes V–X in the presence of MutS (400 nM per monomer – dashed line) or in the absence of protein (solid line). DNA duplexes (concentration 20 nM) contain FRET pair - Alexa-488 (donor) and Alexa-594 (acceptor). The central variable nucleotide pair in DNA is shown in parentheses. The samples were irradiated by light at 470 nm. Spectra were recorded at 500-800 nm. RU - the signal detector in stated units. Each spectrum was recorded at least three times. The figure shows one of the experiments.</p

The plasmid DNAs cleavage by MutH in a MutS-MutL dependent manner.

<p><b>A</b>, Analysis of the G/T-cccDNA treated with MutS-MutL-MutH mixture after 1, 5 or 10 min incubation in 1% agarose gel containing ethidium bromide. The initial cccDNA is shown (0 min). M – DNA ladder. <b>B</b>, Diagram representing the data of hydrolysis by MutS-MutL-MutH mixture of G/T-, G/C-, G/Tg- and A/Tg-cccDNA (the variable nucleotide pair introduced in cccDNA is indicated under the lanes) for 5 min. The experiments were performed 5 times. Error bars are standard deviations of the mean.</p

The general scheme of MutS homodimer interaction with DNA.

<p>The issues investigated in the current work are enclosed in the rectangles.</p

45 bp duplexes V-X containing the variable nucleotide pair and the FRET pair.

<p>Variable nucleotide pair is shown in bold. Alexa-594 (black circle) and Alexa-488 (grey circle) are linked to T residues.The duplexes are obtained by hybridization of three fragments (15-, 17- and 13-mer) on the 45-mer template strand. The nicks in the “bottom” strand of duplexes are indicated by vertical lines.</p

Change in energy transfer efficiency upon MutS binding to different DNA duplexes.

a<p>Variable nucleotide pair.</p><p>The error of the mean indicates a SEM.</p

Characteristics of DNA duplex thermal stabilities.

a<p>Variable nucleotide pair.</p>b<p>Melting temperature of duplex.</p>c<p>Hyperchromic effect.</p>d<p>Concentration per duplex.</p>e<p>Cooperativity of phase transition.</p><p>The averaged data of three experiments are presented. The error of the mean indicates a SD.</p

The MutS interaction with DNA duplexes containing FRET pair.

<p><b>A</b>, Models of MutS localization on DNA relative to fluorophores Alexa-594 (red) and Alexa-488 (green). The central nucleoside pair is indicated under the cartoons. MutS subunit A which interacts with the mismatch specifically is shown in blue-green; subunit B which forms only non-specific contacts with DNA is shown in yellow-red. B and C, Change in fluorescence anisotropy (Δr) upon maximal binding extent of MutS (total concentration per monomer 125 nM) to DNA (20 nM) containing various central nucleotide pairs: B, for Alexa-488; C, for Alexa-594. Error bars are standard deviations of the mean.</p

Parameters of MutS binding to DNA, ATP hydrolysis and nucleotide exchange in ATPase domain of protein.

a<p>Variable nucleotide pair.</p>b<p>The ratio of <i>v</i>₀ of ATP hydrolysis by MutS in the absence or in the presence of duplexes I–IV to <i>v</i>₀ of ATP hydrolysis by MutS in the absence of DNA.</p>c<p>The ratio of rate constant of mant-ADP dissociation from its complex with MutS (<i>k</i>_off^mant-ADP) in the absence or in the presence of duplexes I–IV to <i>k</i>_off^mant-ADP in the absence of DNA.</p><p>The error of the mean indicates a SEM.</p