Base-pair termini

The 5 - 3 exonuclease activity is much less specific in its requirements. A base-paired terminus is all that is required for its activity, irrespective of whether the 5 -terminal end is phosphoryl-ated or not. The 5 - 3 nuclease can remove oligonucleotides up to ten residues long from the 5 -end and the activity of this degrada-tive function is markedly enhanced by concomitant DNA synthesis. It is this property of the enzyme which allows the highly... 

The pattern and efficiencies of strand cleavage at GG steps in duplex DNA reflect the ability of a radical cation to migrate from its initial position through a sequence of base pairs. In an illustrative example, we consider the photochemistry of AQ-DNA(l), which is shown in Fig. 4. AQ-DNA(l) is a 20-mer that contains an AQ group linked to the 5 -end of one strand and has two GG steps in the complementary strand. The proximal GG step is eight base pairs, ca. 27 A, from the 5 -end linked to the AQ, and the distal GG step is 13 base pairs (ca. 44 A) away. The complementary strand is labeled with 32P at its 5 -terminus (indicated by a in Fig. 4). 

Fig. 5 Schematic representation of long distance radical cation migration in DNA. In AQ-DNA(3), irradiation of the anthraquinone group linked at the 5 -terminus leads to reaction at GG steps that are 10, 28, 46 and 55 base pairs from the charge injection site. The solid arrows indicate approximately the amount of reaction observed at each GG step. The plot shows the natural log of the normalized amount of reaction as a function of distance from the AQ. The results appear to give a linear distance dependence...

A structural feature of the T7 DNA which is important in DNA replication is that there is a direct terminal repeat of 160 base pairs at the ends of the molecule. In order to replicate DNA near the 5 terminus, RNA primer molecules have to be removed before replication is complete. There is thus an unreplicated portion of the T7 DNA at the 5 terminus of each strand. The opposite single 3 strands on two separate DNA molecules, being complementary, can pair with these 5 strands, forming a DNA molecule twice as long as the original T7 DNA. The unreplicated portions of this end-to-end bimolecular structure are then completed through the action of... 

Figure 12.5 The structures for four tRNA molecules of yeast, (a) Alanyl-tRNA (b) phenylalanyl-tRNA (c) seryl-tRNA (d) tyrosyl-tRNA. The single letter designations identify the sequence of bases along the single chain. Note that several of these are unusual bases, most of which are methylated (Me). Note also the ACC sequence at the 3 terminus of each tRNA. This is the site to which amino acids are attached in the process of protein synthesis, as indicated. These tRNA molecules have a substantial amount of secondary structure created by formation of Watson-Crick base pairs. Finally, note that the anticoding triplet in the bottom loop is shown.

Sequence 3 examines chain-end effects on DNA dynamics. It contains the coumarin probe one base pair removed from the end of the sequence. This chain end sequence has the same base pair sequence as the normal sequence, but with entire sequence, including the coumarin, shifted towards one terminus. The Stokes shifts from the two sequences are displayed on a relative Stokes shift scale in Fig. 2b. 

DNA polymerases have just one binding site for all four combinations of base pairing—AT, TA, GC, and CG. The specificity of these sites is dictated by the Watson-Crick pairing rules, in that the sites themselves appear to recognize just the overall shape of a correct purine-pyrimidine pair, with the precise specificity resulting from the complementary nature of the base pairing. The polymerase catalyzes the transfer of a complementary deoxynucleoside monophosphate from its triphosphate to the 3 -hydroxyl of the primer terminus (equation 14.1). 

The pol / p/t ddCTP structure shows that, in the absence of a downstream DNA fragment, the lyase domain does not interact with the DNA and is positioned some distance from the active site (Pelletier et al, 1994). However, in a structure solved with gapped DNA, the lyase domain binds to the 5 -phosphate in the DNA gap and interacts with its own carboxy-terminus in the thumb (Sawaya et al, 1997). In common with all polymerases, the fingers subdomain closes down around the correct nucleotide and its complementary template base. This motion corresponds to a 30-degree rotation of a-helix N (the dNTP-binding site) around oc-helix M and thus brings helix N and its bound nucleotide into position to probe correct W-C base pairing. 

The coding region of the type-L isozyme is 2898 base pairs long. It corresponds to 966 amino acid residues with a molecular weight of 109,648, and consists of two regions the amino-terminal extended sequence of 50 residues and the mature protein sequence of 916 residues. The amino acid sequence of the mature protein deduced from the cDNA structure is perfectly matched to the sequence chemically determined as described above. The stop codon, TAA, appeared at position 2942 in the cDNA sequence just after the carboxyl-terminal amino acid of the mature enzyme, indicating that no post-translational processing occurred at the carboxyl terminus. 

The 3 -+5 exonuclease activity plays an important role in polymerization in proof reading the base pair formed at each polymerization step. The enzyme checks the nature of each base-paired primer terminus before the polymerase proceeds to add the next nucleotide to the primer. It thus supplements the capacity of the polymerase to match the incoming nucleotide substrate to the template. A mismatched terminal nucleotide on the primer activates a site on the enzyme which results in the hydrolysis of the phosphodiester bond and the removal of the mismatched residue. The function of this 3 - 5 exonuclease activity is therefore to recognize and cleave incorrectly or non-base paired residues at the 3 -end of DNA chains. It will therefore degrade single stranded DNA and frayed or non-base paired residues at the ends of duplex DNA molecules provided they terminate in a 3 -hydroxyl group. 

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