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Highlights • DNA repair kinetics evaluated in T. brucei nuclear and mitochondrial genomes. • Higher efficiency of DNA repair in T. brucei cells from the mammal than the tsetse. • Differing cell cycle and survival responses to DNA damage in two T. brucei cell types. • Mitochondrial DNA repair is active in T. brucei and can involve RAD51., Genomes are affected by a wide range of damage, which has resulted in the evolution of a number of widely conserved DNA repair pathways. Most of these repair reactions have been described in the African trypanosome Trypanosoma brucei, which is a genetically tractable eukaryotic microbe and important human and animal parasite, but little work has considered how the DNA damage response operates throughout the T. brucei life cycle. Using quantitative PCR we have assessed damage induction and repair in both the nuclear and mitochondrial genomes of the parasite. We show differing kinetics of repair for three forms of DNA damage, and dramatic differences in repair between replicative life cycle forms found in the testse fly midgut and the mammal. We find that mammal-infective T. brucei cells repair oxidative and crosslink-induced DNA damage more efficiently than tsetse-infective cells and, moreover, very distinct patterns of induction and repair of DNA alkylating damage in the two life cycle forms. We also reveal robust repair of DNA lesions in the highly unusual T. brucei mitochondrial genome (the kinetoplast). By examining mutants we show that nuclear alkylation damage is repaired by the concerted action of two repair pathways, and that Rad51 acts in kinetoplast repair. Finally, we correlate repair with cell cycle arrest and cell growth, revealing that induced DNA damage has strikingly differing effects on the two life cycle stages, with distinct timing of alkylation-induced cell cycle arrest and higher levels of damage induced death in mammal-infective cells. Our data reveal that T. brucei regulates the DNA damage response during its life cycle, a capacity that may be shared by many microbial pathogens that exist in variant environments during growth and transmission.

In the past, lattice Boltzmann methods (LBM) have been extensively developed for momentum and energy transport in single-phase and multi-phase fluid systems. Recently, LBM based algorithms have been developed and applied to fundamental Radiative Transport Equations (RTE), including radiation–material interactions and were found very convenient to model radiative energy exchange between radiation and material medium. This work advances the development of Lattice Boltzmann Equations (LBE) for radiative transport by integrating them with existing LBEs for energy and momentum transport for solving multi-physics problems. The multi-physics example problems of thermal energy transport where radiation, conduction and convection all are considered as important modes are modeled via this integrated LBM. These integrated LBM models are used to solve one and two dimensional problems, and highlight the advantage of this approach for solving multi-physics problems in a single framework. First example involves modeling radiative and conductive heat transfer in one-dimensional slab using LBM. The numerical results are compared with existing benchmark P1 solutions. Next example is the simulation of two-dimensional radiative porous burner with hot walls. This problem is simulated with two numerical models: a homogenous porous media and a heterogenous model of packed obstacles which have differential scattering and absorption interactions. The homogenous model uses analytical velocity field and provides a simpler approach, but has limitations in providing detailed analysis. In heterogenous model velocity field, temperature field and radiation field are computed with a set of coupled LBEs. Fluid flowing through heterogenous porous media undergoes conjugate heat exchange with obstacles and also interacts with isotropic incident radiation. These two-dimensional example cases with different material properties are solved with D2Q16 LBE template.

Nuclear DNA replication is, arguably, the central cellular process in eukaryotes, because it drives propagation of life and intersects with many other genome reactions. Perhaps surprisingly, our understanding of nuclear DNA replication in kinetoplastids was limited until a clutch of studies emerged recently, revealing new insight into both the machinery and genome-wide coordination of the reaction. Here, we discuss how these studies suggest that the earliest acting components of the kinetoplastid nuclear DNA replication machinery - the factors that demarcate sites of the replication initiation, termed origins - are diverged from model eukaryotes. In addition, we discuss how origin usage and replication dynamics relate to the highly unusual organisation of transcription in the genome of Trypanosoma brucei.

Trypanosomes are unicellular eukaryotes that cause disease in humans and other mammals. Trypanosoma cruzi and Trypanosoma brucei are the causative agents, respectively, of Chagas disease in the Americas and sleeping sickness in sub-Saharan Africa. To better comprehend the interaction of these parasites with their hosts, understanding the mechanisms involved in the generation of genetic variability is critical. One such mechanism is mismatch repair (MMR), which has a crucial, evolutionarily conserved role in maintaining the fidelity of DNA replication, as well as acting in other cellular processes, such as DNA recombination. Here we have attempted to complement T. brucei MMR through the expression of MSH2 from T. cruzi. Our results show that T. brucei MSH2-null mutants are more sensitive to hydrogen peroxide (H2O2) than wild type cells, suggesting the involvement of MSH2 in the response to oxidative stress in this parasite. This phenotype is reverted by the expression of either the T. cruzi or the T. brucei MSH2 protein in the MSH2-null mutants. In contrast, MMR complementation, as assessed by resistance to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and microsatellite instability, was not achieved by the heterologous expression of T. cruzi MSH2. This finding, associated to the demonstration that mutation of MLH1, another component of the MMR system, did not affect sensitivity of T. brucei cells to H2O2, suggests an additional role of MSH2 in dealing with oxidative damage in these parasites, which may occur independently of MMR.

Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays.

Trypanosome antigenic variation, involving differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres and with DNA recombination. All expressed VSGs are telomeric, and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with eukaryotic telomeres that is directly involved in DNA recombination and in gene silencing. We have tested the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. We show, via the generation of null mutants, that trypanosome Ku is closely involved in telomere length maintenance, more so for a transcriptionally active than an inactive telomere, but exhibits no detectable influence on DNA double strand break repair. The absence of Ku and the consequent great shortening of telomeres had no detectable influence either on the rate of VSG switching or on the silencing of the telomeric promoters of the VSG subset that is expressed in the tsetse fly.

The stable inheritance of ColE1-related plasmids and the normal partition of the E. coli chromosome require the function of the Xer site-specific recombination system. We show that in addition to the XerC recombinase, whose function has already been implicated in this system, a second chromosomally encoded recombinase, XerD, is required. The XerC and XerD proteins show 37% identity and bind to separate halves of the recombination site. Both proteins act catalytically in the recombination reaction. Recombination site asymmetry and the requirement of two recombinases ensure that only correctly aligned sites are recombined. We predict that normal partition of most circular chromosomes requires the participation of site-specific recombination to convert any multimers (arising by homologous recombination) to monomers.