Base-pairing mispairing

Mismatch Repair. Mispairs that break the normal base-pairing rules can arise spontaneously due to DNA biosynthetic errors, events associated with genetic recombination and the deamination of methylated cytosine (Modrich, 1987). With the latter, when cytosine deaminates to uracil, an endonuclease enzyme, /V-uracil-DNA glycosylase (Lindahl, 1979), excises the uracil residue before it can pair with adenine at the next replication. However, 5-methyl cytosine deaminates to form thymine and will not be excised by a glycosylase. As a result, thymine exits on one strand paired with guanine on the sister strand, that is, a mismatch. This will result in a spontaneous point mutation if left unrepaired. For this reason, methylated cytosines form spontaneous mutation hot-spots (Miller, 1985). The cell is able to repair mismatches by being able to distinguish between the DNA strand that exists before replication and a newly synthesized strand. 

An important characteristic of Holliday junctions formed from homologous duplexes is that they can move by a process called branch migration.295 Because of the twofold symmetry of the branched structure the hydrogen bonds of one base pair can be broken while those of a new base pair are formed, the branch moving as shown in Fig. 5-28. Notice that, in this example, the nonhomo-logous (boxed) base pairs TA and GC have become mispaired as TG and AC after branch migration. More significantly, the junction may be cut by a resolvase at the points marked... 

Here the central asterisks designate mispairs and the green shade marks mismatched bases and also the resulting mutant base pair formed in the next replication cycle. 

It is possible for base mispairing in duplex DNA to be corrected by repair mechanisms because the information content is duplicated in the two complementary strands. For example, aberrant base pairing arising from environmental damage such as x-rays, ultraviolet radiation, oxidation, and chemical modification may be repaired as follows. 

Mismatch repair was an essential component of the Holliday model for meiotic recombination in fungi (Holliday, 1965a). This states that recombination depends on the formation of relatively short stretches of hybrid DNA between homologous chromatids within which, if they spanned a region of heterozygosity, a mispaired base-pair would appear. Mismatch repair would determine whether or not aberrant segregations would be found in the tetrads formed on completion of meiosis. 

Although base-pairing specificity confers target selectivity, minimizing the potential for general toxicity, the question of toxicity must be rigorously tested since mispair-ing by a ribozyme to a non-targeted... 

An estimate of the extent of base-pair mismatch between hybridized DNA strands can be made by measuring the thermal stability of the reassociated duplexes. Place hybridized filters in fresh incubation buffer and heat at 5° increments to 90°. At each 5° increase, remove a small sample of the buffer and count. Controls are reassociated homologous DNAs. The amount of nucleotide mispairing has been estimated at 1% for each degree the Tm of the heterologous duplex is lowered (Arm).2 31... 

In all these events, the hydrogen-bonded Watson-Crick base pair is operative and is responsible for DNA reduplication, transcription, and translation. Since mispairing can occur, all these processes are checked for fidelity by several enzymes which can correct for errors [6521. At this and all other levels of DNA reduplication and protein biosynthesis, intermolecular hydrogen bonds between nucleic acids and between nucleic acids and proteins are responsible for recognition, interaction, and, finally, for information transfer. 

There are two mutation processes where mispairing due to enol/imino tautomeric forms could be involved. In one, transition, a purine is replaced by another purine or a pyrimidine by another pyrimidine in the other, transversion, a purine/pyrimidine or pyrimidine/purine exchange takes place. In these mutations, formation of base pairs of the type illustrated in Fig. 20.7 is postulated. 

Figure 3 Kinetic steps during DNA synthesis favor incorporation of correct dNTPs. Most often the DNA polymerase selects the correct dNTP that forms a correct Watson-Crick base pair with the template strand (pathway 1, left). The chemistry of correct dNTP incorporation is rapid, and it allows the polymerase to proceed rapidly to incorporate subsequent dNTPs. The chemistry of incorporating an incorrect dNTP is slow (pathway 2, right), and subsequent elongation of the mispaired 3 terminus is also slow. These two kinetic barriers provide time for the primed template to switch into the proofreading 3 -5 exonuclease active site, where removal of the mispaired 3 terminus is rapid. The excised and fully base paired primed site then switches back to the DNA polymerase active site (dashed arrow).

While considering the presence of U in DNA, it is relevant to point out that this base can also arise in DNA by its misincorporation (instead of T) during normal DNA replication as a consequence of the existence of a small pool of dUTP (1). Not unexpectedly, perturbations that increase the pool size of dUTP relative to that of TTP promote this misincorporation (1). However, the biologic consequences of A U mispairs in DNA are limited because such base pairs have the same coding potential as A T pairs (1). 

In this regard, significant chemical modification is required to effect either keto-enol or amino-imino tautomerism of the nitrogenous bases, with the consequent formation of A C and G T mispairs that fit into the canonical double-helical structure. Notably, the N6-methoxy A C mispairs and the 06-methylated G T mispairs, which are observed in crystalline duplex structures, are isomorphous with standard A T and G C base pairs (Tables 1 and 2) NDB entries bd0009, bdlb26, and bdlb58 (19-21). 

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