Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 64
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This enzyme has an exonuclease activity that can removenucleotides from the 5' end of a base-paired fragment. It is effective with both DNA and RNA. The activity is a 5'3' exonuclease activity. Pol I acts at the nick and displaces it in the 5'3' direction by removing RNAnucleotides one by one and adding DNA nucleotides to the 3' end of the DNA strand. When all of the RNAnucleotides have been removed, DNA ligase joins the 3'-OH group to the terminal 5'-P of the precursor fragment.By this sequence of events, the precursor fragment is assimilated into the lagging strand.
When the next precursorfragment reaches the RNA primer of the fragment just joined, the sequence begins again. Polymerase I is essentialfor DNA replication, because the 5'-to-3' exonuclease activity required for removing the RNA primer and joiningthe precursor fragments is not present in Pol III.Other Proteins Needed for DNA ReplicationThe roles of some of the most important components of DNA replication are depicted in Figure 5.27. Thereplication process includes, in addition to theFigure 5.23Differences between DNA and RNA. The chemical groups that are highlighted are thedistinguishing features of deoxyribose and ribose and of thymine and uracil.Page 197Figure 5.24Priming of DNA synthesis with an RNA segment.
The DNA template strand isshown in blue; newly synthesized DNA is shown in beige. The RNA segment,which was made by an RNA polymerase (primase), is shown in green. In the firststep, DNA polymerase adds a deoxyribonucleotide to the 3' end of the primer.Successive deoxyribonucleotides are added to the 3' end of the growing chain.Figure 5.25Prior to their being joined, each precursor fragment in the lagging strandhas the structure shown here.
The short RNA primer is shown ingreen.Page 198Figure 5.26Sequence of events in the joining of adjacent precursor fragments. (A) The DNA polymerase of theupstream fragment meets the RNA primer from the next precursor fragment down-stream. (B) The RNAnucleotide immediately adjacent is excised and replaced with a DNA nucleotide. (C) EachRNA nucleotide is cleaved and replaced in turn. (D) When all the RNA nucleotides have been replaced,the adjacent ends of the DNA strand are joined.polymerase III complex and the RNA primase complex, at least one type of topoisomerase, helicase proteins thatbind at the replication fork and unwind the double helix, single-strand binding proteins that bind with andstabilize the single-stranded DNA at the replication fork, the DNA ligase that joins DNA fragments, and thepolymerase I complex (not shown in Figure 5.27) that eliminates the RNA primers from precursor fragmentsbefore joining can take place.Page 199Figure 5.27Role of some of the key proteins in DNA replication.
The DNA polymerase III complex and the primase complex are both composedof multiple different polypeptide subunits. DNA polymerase I, which joins precursor fragments where they meet, is not shown.5.7—The Isolation and Characterization of Particular DNA FragmentsThis section and the following sections show how our knowledge of DNA structure and replication has been put topractical use in the development of procedures for the isolation and manipulation of DNA. One set of procedures isbased on the ability of duplex DNA to be separated into single strands that, under the proper conditions, can formduplexes with other single strands having complementary sequences.
This phenomenon is described next.Denaturation and RenaturationThe double-stranded helical structure of DNA is maintained by forces that include hydrogen bonds between thebases of complementary pairs. When solutions of DNA are exposed to temperatures considerably higher than thosenormally encountered in most living cells or to excessively high pH, the hydrogen bonds break and the pairedstrands separate. Unwinding of the helix happens in less than a few minutes, the time depending on the length ofthe molecule.
When the helical structure of DNA is disrupted and the strands are separated, the molecule is said tobe denatured. A common way to detect denaturation is by measuring the capacity of DNA in solution to absorbultraviolet light of wavelength 260 nm. The absorption at 260 nm (A260) of a solution of single-stranded moleculesis 37 percent higher than the absorption of the double-stranded molecules at the same concentration.
When a DNAsolution is slowly heated and the value of A260 is recorded at various temperatures, a curve called a melting curveis obtained. An example is shown in Figure 5.28. The melting transition is usually described in terms of thetemperature at which the increase in the value of A260 is half complete. This temperature is called the meltingtemperature and is denoted by Tm.Page 200Figure 5.28A melting curve of DNA showing the Tm and possible shapes of a DNA moleculeat various degrees of denaturation.The value of Tm increases with G + C content, because G - C pairs, joined by three hydrogen bonds, are strongerthan A - T pairs, which are joined by two hydrogen bonds.The single strands in a solution of denatured DNA can, under certain conditions, re-form double-stranded DNA.This process is called renaturation or annealing.
For renaturation to happen; two requirements must be met: (1)The salt concentration must be high (>0.25 M) to neutralize the negative charges of the phosphate groups, whichwould otherwise cause the complementary strands to repel one another; and (2) the temperature must be highenough to disrupt hydrogen bonds that form at random between short sequences of bases within the same strand,but not so high that stable base pairs between the complementary strands would be disrupted. A temperature about20°C below Tm is usually optimal. The initial phase of renaturation is a slow process, because its rate is limited bythe random chance that a region of two complementary strands will come together to form a short sequence ofcorrect base pairs. This initial pairing step is followed by a rapid pairing of the remaining complementary bases andrewinding of the helix.
Rewinding is accomplished in a matter of seconds, and its rate is independent of DNAconcentration (because the complementary strands have already found each other). Correct initial base pairing ofall molecules in a sample is concentration-dependent and may require several minutes to many hours whenstandard conditions are used.An example utilizing a hypothetical DNA molecule will enable us to understand some of the molecular details ofrenaturation. Consider the double-stranded DNA molecule in Figure 5.29A containing 30,000 base pairs, in whicha specific sequence of six base pairs appears only twice.
(In general, a six-base sequence would be expected to befound an average of about seven times in a molecule of this length.) This molecule is heat-denatured (Figure5.29B) and then the temperature is lowered to promote renaturation. In the solution of these denatured molecules, arandom collision between noncomplementary base sequences cannot initiate renaturation, but a collision thatbrings together sequences I and II' or I' and II can result in base pairing (Figure 5.29C).
This pairing will betransient at the elevated temperatures used for renaturation, because the paired region is short and the adjacentbases in the two strands are out of register and unable to pair, as is required to form a double-stranded molecule.On the other hand, if a collision results in the pairing of sequence I with I' or II with II'—or any other shortcomplementary sequences—then pairing of the adjacent bases (and all other bases in the strands) will proceed in azipperlike action (Figure 5.29D).
The main point is that only base pairing that brings the complementary sequencesinto register will cause renaturation.Nucleic Acid HybridizationAnother important point is that because a solution of denatured DNA usually contains a large number of identicalDNA molecules, a double-stranded molecule formed by renaturation is rarely composed of the same two strandsthat were paired beforePage 201Figure 5.29Denaturation and renaturation of a DNA duplex. (A) Original duplex. (B) High temperature causes the strands to come apartLowering the temperature allows short complementary stretches to come together.
(C) If the sequences flanking the paired regionare not complementary, then the pairing is unstable and the strands come apart again. (D) If the sequences flanking thepaired region are complementary, then further base pairing stabilizes the renatured duplex.Page 202renaturation. This phenomenon is the basis of one of the most useful methods in molecular genetics—nucleic acidhybridization. In this context, molecular hybrids are molecules formed when two single nucleic acid strandsobtained by denaturing DNA from different sources have sufficient sequence complementarity to form a duplex.An example of the use of DNA-DNA hybridization is determination of the fraction of DNA in two differentspecies that have common base sequences.
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