RecQ helicases in the malaria parasite Plasmodium falciparum affect genome stability, gene expression patterns and DNA replication dynamics.

Antoine Claessens,Yvon Sterkers,Lia Chappell,Adam Stanton,Lynne M Harris,Catherine J Merrick,Julian C Rayner,Pamela Veneziano-Broccia,Nada Kuk,Slavica Stanojcic

doi:10.1371/journal.pgen.1007490

Abstract

The malaria parasite Plasmodium falciparum has evolved an unusual genome structure. The majority of the genome is relatively stable, with mutation rates similar to most eukaryotic species. However, some regions are very unstable with high recombination rates, driving the generation of new immune evasion-associated var genes. The molecular factors controlling the inconsistent stability of this genome are not known. Here we studied the roles of the two putative RecQ helicases in P. falciparum, PfBLM and PfWRN. When PfWRN was knocked down, recombination rates increased four-fold, generating chromosomal abnormalities, a high rate of chimeric var genes and many microindels, particularly in known ‘fragile sites’. This is the first identification of a gene involved in suppressing recombination and maintaining genome stability in Plasmodium. By contrast, no change in mutation rate appeared when the second RecQ helicase, PfBLM, was mutated. At the transcriptional level, however, both helicases evidently modulate the transcription of large cohorts of genes, with several hundred genes—including a large proportion of vars—showing deregulated expression in each RecQ mutant. Aberrant processing of stalled replication forks is a possible mechanism underlying elevated mutation rates and this was assessed by measuring DNA replication dynamics in the RecQ mutant lines. Replication forks moved slowly and stalled at elevated rates in both mutants, confirming that RecQ helicases are required for efficient DNA replication. Overall, this work identifies the Plasmodium RecQ helicases as major players in DNA replication, antigenic diversification and genome stability in the most lethal human malaria parasite, with important implications for genome evolution in this pathogen.

Highlights

Protozoan Plasmodium parasites are the causative agents of human malaria, a disease responsible for widespread morbidity and almost half a million deaths each year [1]
Human malaria is caused by Plasmodium parasites, with most of the mortality being caused by one species, Plasmodium falciparum
This parasite has an unusual genome: it is exceptionally biased towards A and T nucleotides rather than G and C, and it contains specific areas rich in hypervariable virulence-associated genes which evolve very rapidly to produce new variants. This evolution is probably vital for the parasite to evade the human immune system and maintain chronic infections, but how it is controlled at a molecular level remains unknown

Summary

Introduction

Protozoan Plasmodium parasites are the causative agents of human malaria, a disease responsible for widespread morbidity and almost half a million deaths each year [1]. P. falciparum has one of the most highly A/T-biased genomes ever sequenced, at ~81% A/T [2] This is maintained by a high mutational bias towards G/C to A/T transitions [3] and the resultant genome contains a preponderance of A/T repeat tracts [4]. ~80 putative G-quadruplex (G4) forming sequences (non-double-helical structures that require four closely-spaced tracts of at least three guanines to form [7]) are found outside the intrinsically guanine-rich telomeres in P. falciparum [8, 9] Both hairpins and G4s in DNA are implicated in stalling RNA and DNA polymerases, and in promoting recombination events via DNA breakage at stalled replication forks [10, 11]

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