Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes

Abstract

ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd ؉ bacterium as the result of replication errors or recombinationdependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with Ϸ600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.Within a bacterium that has a classical restriction and modification (R-M) system, the nucleotide sequences that define the targets for attack by the resident restriction endonuclease are concealed by the modification of appropriate bases within them. For some systems this modification is achieved by the methylation of specific adenine residues, and for others it is achieved by methylation of cytosine residues. The restriction endonuclease has the potential to attack DNA from different strains of the same species because foreign DNA generally lacks the protective imprint of the relevant methyltransferase (for reviews see refs. 1 and 2). Restriction of the host cell's newly synthesized DNA normally is avoided, because the unmethylated strand of each target sequence produced by DNA replication is methylated before the next round of replication. If, however, resident DNA were to acquire unmodified target sequences, would it, like foreign DNA, become a substrate for restriction? In this paper we show that in situations where the modification of the host DNA by a type I R-M system fails, an alternative level of protection impairs the endonuclease activity of the restriction system and the bacteria survive.A type I R-M system is encoded by three genes: hsdR, hsdM, and hsdS. The three polypeptides, HsdR, HsdM, and HsdS, often designated R, M, and S, assemble to give an enzyme (R 2 M 2 S 1 ) that modifies hemimethylated DNA and restricts unmethylated DNA. A smaller complex (M 2 S 1 ) has only the methyltransferase activity. The S subunit confers target specificity; hence, both complexes and both activities respond to the same nucleotide sequence.Type

Immigration control of DNA in bacteria: self versus non-self

“…The expected consequence of this mutation in vivo would be fragmentation of the bacterial chromosome. However, recent experiments contradict this expectation (Makovets et al, 1999 ;Doronina & Murray, 2001 ;Cromie & Leach, 2001). It would appear that when modification fails, the bacterial cell is endowed with the means of causing the restriction pathway to abort before the enzymes break the DNA.…”

“…10), or directly by mutations that create target sequences. The original genetic evidence for the creation of vulnerable target sequences by mutation (Makovets et al, 1999) is now supported by the demonstration of breaks in the bacterial chromosome when E. coli K-12 is treated with 2-AP. The breaks are dependent on EcoKI and, as predicted if they arise by base substitutions, their generation requires two rounds of replication (Cromie & Leach, 2001).…”

“…A similar response has been demonstrated for a variety of agents that damage DNA, including mutagens such as the base analogue 2-aminopurine (2-AP), and defects in some genes that affect DNA metabolism, e.g. dam, topA and mutD (dnaQ) (Efimova et al, 1988a, b ;Thoms & Wackernagel, 1984 ;Makovets et al, 1999). DNA damage may generate unmodified target sequences as a consequence of the repair of double-strand breaks by homologous recombination (see Fig.…”

“…One simple solution is to delay production of the restriction enzyme until the modification enzyme has had time to modify all the targets in the bacterial chromosome . This process, however, takes many generations following the acquisition of the genes specifying EcoKI, because unmethylated DNA is a very poor substrate for modification (Makovets, 1999). For type II R-M systems, transcriptional control of gene expression is well documented (see Raleigh & Brooks, 1998), but transcriptional control has not been found to be relevant for any type I or type III system that has been investigated (Loenen et al, 1987 ;Kulik & Bickle, 1996 ;Redaschi & Bickle, 1996).…”

“…Some data will challenge our long-established belief that modification of DNA is essential to distinguish whether the DNA is ' self or foreign '. Experiments show that while modification of DNA is sufficient, it is not always essential to identify resident DNA as self (Makovets et al, 1999 ;Doronina & Murray, 2001). …”