Abstract
Ultraviolet (UV) irradiation is not known to induce chromosomal fragmentation in sublethal doses, and yet UV irradiation causes genetic instability and cancer, suggesting that chromosomes are fragmented. Here we show that UV irradiation induces fragmentation in sublethal doses, but the broken chromosomes are repaired or degraded by RecBCD; therefore, to observe full fragmentation, RecBCD enzyme needs to be inactivated. Using quantitative pulsed field gel electrophoresis and sensitive DNA synthesis measurements, we investigated the mechanisms of UV radiation-induced chromosomal fragmentation in recBC mutants, comparing five existing models of DNA damage-induced fragmentation. We found that fragmentation depends on active DNA synthesis before, but not after, UV irradiation. At low UV irradiation doses, fragmentation does not need excision repair or daughter strand gap repair. Fragmentation absolutely depends on both RecA-catalyzed homologous strand exchange and RuvABC-catalyzed Holliday junction resolution. Thus, chromosomes fragment when replication forks stall at UV lesions and regress, generating Holliday junctions. Remarkably, cells specifically utilize fork breakage to rescue stalled replication and avoid lethality.
Highlights
Ultraviolet (UV) light causes genetic instability, suggesting chromosome fragmentation
Through our interest in low level spontaneous chromosomal fragmentation induced by endogenous DNA damage and following the lead of Michel et al [39], we developed a sensitive technique based on pulsed field gel electrophoresis to detect and quantify chromosomal fragmentation in E. coli [31, 40]
When we blocked the repair and degradation of double strand ends with the recBC defect, we found that the same dose of UV irradiation (36 J/m2) followed by identical growth conditions fragments up to 30% of the chromosomal DNA (Fig. 1, A and B), suggesting that the low levels of fragmentation in the wild type cells are due to the efficient repair of double strand breaks and to linear DNA degradation, the two processes catalyzed by the RecBCD enzyme [43], and that RecBCD needs to be saturated to reveal fragmentation
Summary
Ultraviolet (UV) light causes genetic instability, suggesting chromosome fragmentation. Results: UV light induces chromosomal fragmentation, which is independent of excision of UV lesions but requires replication forks, homologous strand exchange, and Holliday junction resolution. Of Microbiology, University of Illinois at Urbana-Champaign, B103 C&LSL, 601 S. We hypothesized that sublethal UV irradiation doses trigger genetic instability by inducing chromosomal fragmentation, which avoided previous detection in wild type cells [18, 19] because of efficient double strand break repair or linear DNA degradation. When we used our sensitive assay to measure chromosome instability in E. coli after UV irradiation, we found highly fragmented chromosomes Genetic analysis of this fragmentation in combination with the sensitive measurements of the DNA synthesis rate ruled out all the current models of DNA damage-induced fragmentation except the one in which stalled replication forks. UV Radiation-induced Chromosomal Fragmentation actively regress to form Holliday junctions, which are resolved to break the forks
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