News flash: life was just discovered on Mars scientists have begun to examine Ma
ID: 85657 • Letter: N
Question
News flash: life was just discovered on Mars scientists have begun to examine Martian life and find that it is cellular, and probabaly driven from an early meteorite-delivered colonization event from the Earth when the planets were both still young .analysis of Martian cell reveals that Mars life uses the same DNA and RNA that we do, but all the enzyme catalysts are also made of nucleic acids, rather than of proteins. you find the Martian equivalent to the Terran protein restriction endonuclease Sau 3ai.describe the chemical means by which this enzyme is likely to recognise its substrate with high specificity. a draw would helpExplanation / Answer
A) Here, we report that Sau3AI, an unusually large type II restriction enzyme with sequence homology to the mismatch repair protein MutH,5 Structural similarities in the N- and C-terminal halves of the protein suggest that Sau3AI is a pseudo-dimer, i.e. a polypeptide with two similar domains. Since Sau3AI displays a nonlinear dependence of cleavage activity on enzyme concentration and a strong preference for substrates with two recognition sites over those with only one, it is likely that the functionally active form of Sau3AI is a dimer of a pseudo-dimer. Indeed, electron microscopy studies demonstrate that two distant recognition sites are brought together through DNA looping induced by the simultaneous binding of twoSau3AI molecules to the DNA. We suggest that the dimeric form of Sau3AI supplies two DNA-binding sites, one that is associated with the catalytic center and one that serves as an effector site.
B) Orthodox type II restriction endonucleases are homo dimeric enzymes that cleave their substrates within or directly adjacent to their recognition sequence. It became increasingly apparent over the last years that several variations to this theme exist, namely in the form of the monomeric type IIs restriction endonucleases that dimerize on the DNA substrate via their catalytic domains, e.g. FokI; the dimeric type IIe enzymes, e.g. EcoRII and NaeI, which require binding to a second recognition site for cleavage; dimeric restriction endonucleases like Sgr AI, which also require two recognition sites for activity and tetramerize on the DNA; and tetrameric type IIf restriction endonucleases like SfiI, Cfr10I, and NgoMIV), which interact with two recognition sites, loop out the DNA, and cleave the two sites in a concerted manner. Structural information is available for FokI, NaeI, Cfr10I, and NgoMIV, in addition to structural information on the orthodox homo dimeric type II restriction endonucleases BamHI, BglI,BglII, BsoBI, EcoRI, EcoRV,MunI, and PvuII. The typical restriction endonuclease fold is also found in endonucleases involved in DNA repair and Vsr endonuclease recombination T7 endonuclease I and -exonuclease, and transposition.
C) It was interesting to note that the type II restriction endonucleaseSau3AI shares sequence homology with the DNA mismatch repair endonuclease MutH. Both Sau3AI and MutH recognize the same DNA sequence (GATC); but whereas MutH nicks the un methylated strand of a hemimethylated or un methylated GATC site, Sau3AI cleaves both strands of GATC sites regardless of the methylation status. It shows only catalytic activity after mismatch-dependent activation by MutS and MutL, whereas in vitro, the requirement for MutS and MutL is less pronounced. In contrast to MutH, however, Sau3AI is an active enzyme by itself. The quaternary structure of Sau3AI is not known, but it is likely that it is a functional dimer, as most type II restriction endonucleases that have to catalyze a double-strand cut.Sau3AI is an unusually large restriction endonuclease. It possesses an additional 270 residues C-terminal to the presumptive catalytic N-terminal domain which is homologous in sequence to MutH, resulting in its being twice the size of MutH. Therefore, the question arises concerning the function of this additional C-terminal domain. It has been speculated that the role of these residues inSau3AI may be the functional equivalent to that of MutL and MutS in the case of MutH, such that Sau3AI is permanently activated.
D) Since there is only a small amount of information regarding the biochemistry of Sau3AI, we therefore started to investigate some of the properties of Sau3AI, namely its quaternary structure and its mode of DNA cleavage. Here, we provide evidence thatSau3AI defines a new subtype of type II restriction endonucleases, being a monomer in solution that, to become active, has to dimerize in the presence of DNA, probably by binding to two recognition sites, which is accompanied by DNA looping. We conclude this from the following findings. First, we show thatSau3AI, unlike most other type II restriction endonucleases, is a monomer in solution. Second, the rate of DNA cleavage bySau3AI is not linearly proportional to the protein concentration, suggesting a requirement for cooperative binding ofSau3AI to DNA to afford cleavage. Third, substrates containing more than one GATC site are cleaved by an order of magnitude more quickly than substrates with a single GATC site, suggesting either that a monomer of Sau3AI is interacting with two GATC sites or, more likely, that Sau3AI dimerizes on the DNA, in response to binding to two GATC sites, a conclusion that is substantiated by electron microscopy, which shows thatSau3AI induces loops on DNA with two GATC sites.