The Anionic Phospholipids in the Plasma Membrane Play an Important Role in Regulating the Biochemical Properties and Biological Functions of RecA Proteins

Abstract

Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during normal growth, which are redistributed along the filamented cell axis upon induction of the SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby playing an important role in RecA-dependent homologous recombination. In addition, in vivo and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids. However, there have been almost no data on the association of mycobacterial RecA proteins with the plasma membrane and the effects of membrane components on their function. Here, we show that mycobacterial RecA proteins specifically interact with phosphatidylinositol and cardiolipin among other anionic phospholipids; however, they had no effect on the ability of RecA proteins to bind single-stranded DNA. Interestingly, phosphatidylinositol and cardiolipin impede the DNA-dependent ATPase activity of RecA proteins, although ATP binding is not affected. Furthermore, the ability of RecA proteins to promote DNA strand exchange is not affected by anionic phospholipids. Strikingly, anionic phospholipids suppress the RecA-stimulated autocatalytic cleavage of the LexA repressor. The Mycobacterium smegmatis RecA foci localize to the cell poles during normal growth, and these structures disassemble and reassemble into several foci along the cell after the induction of DNA damage. Taken together, these data support the notion that the interaction of RecA with cardiolipin and phosphatidylinositol, the major anionic phospholipids of the mycobacterial plasma membrane, may be physiologically relevant, as they provide a scaffold for RecA storage and may regulate recombinational DNA repair and the SOS response.

UvrA and UvrC subunits of the Mycobacterium tuberculosis UvrABC excinuclease interact independently of UvrB and DNA

“…The western blot assay was performed as previously described . Protein samples (15 μL) were separated by SDS/PAGE, transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk in Tris‐buffered saline and probed with either anti‐MtUvrB, anti‐MtUvrC or anti‐His antibodies at dilution 1 : 5000.…”

Few-Layer Black Phosphorus as an Artificial Substrate for DNA Replication

“…The smear or wrong bands located outside the target band position come from nonspecific amplification. 22 We found that new DNA strands with a length of 689 bp were successfully synthesized. Both BP and Pfu complexes of physical adsorption or chemical bonding could enhance the DNA replication efficiency (Figure 2c).…”

“…The activated initiator protein DnaA at the cell membrane recognizes and binds the oriC site of the bacterial chromosome to initiate DNA replication. , When anionic phospholipids are depleted, the chromosomal replication of E. coli is inhibited. Although a lot of effort has been expended to investigate the DNA/membrane or polymerase/membrane complex of the bacteria, it is still unclear how the membrane assists in DNA replication. − …”

The intrinsic ATPase activity of Mycobacterium tuberculosis UvrC is crucial for its damage‐specific DNA incision function

“…The assay was carried out as described previously [37,67,68]. Briefly, the proteins from SDS/PAGE gels were transferred onto ethanol‐treated polyvinylidene difluoride membranes in a transfer buffer [20 m m Tris/HCl, pH 8, 190 m m glycine and 20% methanol] at 100 V for 1 h at 4 °C.…”

“…The assay was performed as described [67]. The reaction buffer containing 25 m m Tris/HCl (pH 7.5), 10 m m MgCl 2 , 2.5 m m DTT, 200 pmol [α‐ 32 P]ATP and increasing amounts of MtUvrC or EcUvrC was incubated at 37 °C.…”