Complementary DNA strands

DNA repair is possible largely because the DNA molecule consists of two complementary strands. DNA damage in one strand can be removed and accurately replaced by using the undamaged complementary strand as a template. We consider here the principal types of repair systems, beginning with those that repair the rare nucleotide mismatches that are left behind by replication. 

Lambert P.F., Kawashima E., ReznikoffW.S. (1987). Secondary structure at the bacteriophage G4 origin of complementary strand DNA synthesis in vivo requirements. Gene 53 257-264. 

Lee G U, Chrisey L A and Colton R J 1994 Direct measurement of the forces between complementary strands of DNA Science 266 771... 

Dou ble hel ix (Section 28 8) The form in which DNA normally occurs in living systems Two complementary strands of DNA are associated with each other by hydrogen bonds be tween their base pairs and each DNA strand adopts a helical shape... 

Artificial endonucleases, ie, molecules able to cleave double-stranded DNA at a specific sequence, have also been developed. These endonucleases can be obtained by attaching a chemically reactive group to a sequence-specific oligonucleotide. When the oligonucleotide is bound to its complementary sequence, the activation of the reactive group results in double-stranded DNA cleavage. 

Cellular protein biosynthesis involves the following steps. One strand of double-stranded DNA serves as a template strand for the synthesis of a complementary single-stranded messenger ribonucleic acid (mRNA) in a process called transcription. This mRNA in turn serves as a template to direct the synthesis of the protein in a process called translation. The codons of the mRNA are read sequentially by transfer RNA (tRNA) molecules, which bind specifically to the mRNA via triplets of nucleotides that are complementary to the particular codon, called an anticodon. Protein synthesis occurs on a ribosome, a complex consisting of more than 50 different proteins and several stmctural RNA molecules, which moves along the mRNA and mediates the binding of the tRNA molecules and the formation of the nascent peptide chain. The tRNA molecule carries an activated form of the specific amino acid to the ribosome where it is added to the end of the growing peptide chain. There is at least one tRNA for each amino acid. 

Another class of DNA-binding proteins are the polymerases. These have a nonspecific interaction with DNA because the same protein acts on all DNA sequences. DNA polymerase performs the dual function of DNA repHcation, in which nucleotides are added to a growing strand of DNA, and acts as a nuclease to remove mismatched nucleotides. The domain that performs the nuclease activity has an a/P-stmcture, a deep cleft that can accommodate double-stranded DNA, and a positively charged surface complementary to the phosphate groups of DNA. The smaller domain contains the exonuclease active site at a smaller cleft on the surface which can accommodate a single nucleotide. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Section 28.14 The nucleotide sequence of DNA can be deter-mined by a technique in which a short section of single-stranded DNA is allowed to produce its complement in the presence of dideoxy analogs of ATP, TTP, GTP, and CTP DNA for-mation ter-minates when a dideoxy analog is incorporated into the growing polynucleotide chain. A mixture of polynucleotides differing from one another by an incremental nucleoside is produced and analyzed by electrophoresis. From the observed sequence of the complementary chain, the sequence of the original DNA is deduced. 

FIGURE 12.1 DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule. Each original strand of the double helix serves as a template, and the sequence of nucleotides in each of these strands is copied to form a new complementary strand by the enzyme DNA polymerase. By this process, biosynthesis yields two daughter DNA duplexes from the parental double helix. 

If cis the concentration of single-stranded DNA at time t, then the second-order rate equation for two complementary strands coming together is given by the rate of decrease in c ... 

Examine DNA. How many base pairs does it contain Starting from one end, write down the sequence of bases in one strand. Write down the sequence in the complementary strand. Is this a proper DNA fragment, or does it contain base-pair mismatches ... 

Predicting the Complementary Base Sequence in Double-Stranded DNA... 

Figure 28.4 A representation of semiconservative DNA replication. The original double-stranded DNA partially unwinds, bases are exposed, nucleotides line up on each strand in a complementary manner, and two new strands begin to grow. Both strands are synthesized in the same 5 - 3 direction, one continuously and one in fragments.

The double-stranded DNA to be amplified is heated in the presence of Taq polymerase, Mg2+ ion. the four deoxynucleotide triphosphate monomers (dNTPs), and a large excess of two short oligonucleotide primers of about 20 bases each. Each primer is complementary to the sequence at the end of one of the target DNA segments. At a temperature of 95 °C, double-stranded DNA denatures, spontaneously breaking apart into two single strands. 

Transcription (Section 28.4) The process by which the genetic information encoded in DNA is read and used to synthesize RNA in the nucleus of the cell. A smal I portion of double-stranded DNA uncoils, and complementary ribonucleotides line up in the correct sequence for RNA synthesis. 

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