Escherichia coli strains lacking protein HU are UV sensitive due to a role for HU in homologous recombination

Abstract

hupA and hupB encode the alpha and beta subunits of the Escherichia coli histone-like protein HU. Here we show that E. coli hup mutants are sensitive to UV in the rec+ sbc+, recBC sbcA, recBC sbcBC, umuDC, recF, and recD backgrounds. However, hupAB mutations do not enhance the UV sensitivity of resolvase-deficient recG ruvA strains. hupAB uvrA and hupAB recG strains are supersensitive to UV. hup mutations enhance the UV sensitivity of ruvA strains to a much lesser extent but enhance that of rus-1 ruvA strains to the same extent as for rus+ ruv+ strains. Our results suggest that HU plays a role in recombinational DNA repair that is not specifically limited to double-strand break repair or daughter strand gap repair; the lack of HU affects the RecG RusA and RuvABC pathways for Holliday junction processing equally if the two pathways are equally active in recombinational repair; the function of HU is not in the substrate processing step or in the RecFOR-directed synapsis action during recombinational repair. Furthermore, the UV sensitivity of hup mutants cannot be suppressed by overexpression of wild-type or mutant gyrB, which confers novobiocin resistance, or by different concentrations of a gyrase inhibitor that can increase or decrease the supercoiling of chromosomal DNA.

FIG. 1

Effects of hup mutations in different genetic backgrounds on sensitivity to UV. Strains were grown in LB to log phase (OD₆₀₀ ≈ 0.5), diluted in 1% NaCl, and irradiated on the surface of LB agar plates. Surviving colonies were scored after 24 h of incubation in the dark. The strains identified by genotype were JR1669, JR1670, JR1671, JR1672, and JR1672 transformed with plasmid pYK20 (a), JC7623, SL1012, SL1013, and SL1014 (b), and JC8679, SL1015, SL1016, and SL1017 (c).

FIG. 2

Interactions in terms of UV sensitivity between hup and other genes. Strains were grown in LB to log phase (OD₆₀₀ ≈ 0.5), diluted in 1% NaCl, and irradiated on the surface of LB agar plates. Surviving colonies were scored after 24 h of incubation in the dark. The strains identified by genotype were BH200, SL1022, SL1023, and SL1024 (a), SL1018, SL1019, SL1020, and SL1021 (b), SL1037, SL1038, SL1039, and SL1040 (c), AQ9947, SL1031, SL1032, and SL1033 (d), N2057, SL1034, SL1035, and SL1036 (e), TNM759, SL1041, SL1042, and SL1043 (f), BT125, SL1028, SL1029, and SL1030 (g), and N1234, SL1025, SL1026, and SL1027 (h).

FIG. 3

Effects of the DNA gyrase inhibitor novobiocin on the UV sensitivity of wild-type (JR1669; ♦) and hupAB (JR1672; ∗) strains. Strains were grown in LB to log phase (OD₆₀₀ ≈ 0.5), diluted in 1% NaCl, and spread on the surface of LB agar plates containing 0 to 60 μg of novobiocin per ml. Surviving colonies were scored after 48 h of incubation in the dark. (a) Survival curves of unirradiated cells; (b) survival curves of UV-irradiated cells (the survival fractions are expressed as those obtained from the UV-irradiated plates divided by those obtained from the unirradiated plates containing the corresponding concentrations of novobiocin).