Genome Replication. Topological Problems. Mitesh Shrestha

Transcription

1 Genome Replication Topological Problems Mitesh Shrestha

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3 Watson-Crick scheme for DNA replication

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5 Topological problem and its solution The most trivial of difficulties during DNA replication is the possibility of the daughter molecules getting tangled up. More critical is the rotation that would accompany the unwinding: with one turn occurring for every 10 bp of the double helix, complete replication of the DNA molecule in human chromosome 1, which is 250 Mb in length, would require 25 million rotations of the chromosomal DNA. It is difficult to imagine how this could occur within the constrained volume of the nucleus, but the unwinding of a linear chromosomal DNA molecule is not physically impossible. In contrast, a circular double-stranded molecule, for example a bacterial or bacteriophage genome, having no free ends, would not be able to rotate in the required manner and so, apparently, could not be replicated by the Watson-Crick scheme.

6 Failed Solutions The difficulty relates to the plectonemic nature of the double helix, this being the topological arrangement that prevents the two strands of a coil being separated without unwinding. The problem would therefore be resolved if the double helix was in fact paranemic, because this would mean that the two strands could be separated simply by moving each one sideways without unwinding the molecule. It was suggested that the double helix could be converted into a paranemic structure by supercoiling in the direction opposite to the turn of the helix itself, or that within a DNA molecule the right-handed helix proposed by Watson and Crick might be balanced by equal lengths of a left-handed helical structure. The possibility that double-stranded DNA was not a helix at all, but a side-by-side ribbon structure, was also briefly considered, this idea surprisingly being revived in the late 1970s and receiving a rather acerbic response from Crick and his colleagues. Each of these proposed solutions to the topological problem were individually rejected for one reason or another, most of them because they required alterations to the double helix structure, alterations that were not compatible with the X-ray diffraction results and other experimental data pertaining to DNA structure.

7 General Terms Linking number ( L k) : The number of Watson-Crick twists found in a circular chromosome in a (usually imaginary) planar projection. This number is physically "locked in" at the moment of covalent closure of the chromosome, and cannot be altered without strand breakage. The linking number defines the number of times a strand of DNA winds in the right-handed direction around the helix axis when the axis is constrained to lie in a plane

8 Twist (Tw): The number of Watson-Crick twists in the chromosome when it is not constrained to lie in a plane. If one goes around the superhelically twisted chromosome, counting secondary Watson-Crick twists, that number will be different from the number counted when the chromosome is constrained to lie flat. In general, the number of secondary twists in the native, supertwisted chromosome is expected to be the "normal" Watson-Crick winding number, meaning a single 10-base-pair helical twist for every 34 Å of DNA length. Twist is a measure of the helical winding of the DNA strands around each other. Given that DNA prefers to form B-type helix, the preferred twist = number of basepair/10.

9 Wr, called "writhe", is the number of superhelical twists. Since biological circular DNA is usually underwound, L k will generally be less than Tw, which means that Wrwill typically be negative. Writhe is a measure of the coiling of the axis of the double helix. A right-handed coil is assigned a negative number (negative supercoiling) and a left-handed coil is assigned a positive number (positive supercoiling) L=T+W For example, in the circular DNA of 5400 basepairs, the linking number is 5400/10=540 If no supercoiling, then W=0, T=L=540

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13 The correct solution to topology problem The first real progress towards a solution of the topological problem came in 1954 when Delbrück proposed a breakage-and-reunion model for separating the strands of the double helix (Holmes, 1998). In this model, the strands are separated not by unwinding the helix with accompanying rotation of the molecule, but by breaking one of the strands, passing the second strand though the gap, and rejoining the first strand. This scheme is in fact very close to the correct solution to the topological problem, being one of the ways in which DNA topoisomerases work

14 DNA topoisomerases and its types Topoisomerases are enzymes that regulate the overwinding or underwinding of DNA. In order to prevent and correct these types of topological problems caused by the double helix, topoisomerases bind to either single-stranded or double-stranded DNA and cut the phosphate backbone of the DNA. This intermediate break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed again. Since the overall chemical composition and connectivity of the DNA do not change, the tangled and untangled DNAs are chemical isomers, differing only in their global topology, thus their name. Topoisomerases are isomerase enzymes that act on the topology of DNA.

15 Types of Topoisomerases Topoisomerases can fix these topological problems and are separated into two types depending on the number of strands cut in one round of action. Both these classes of enzyme utilize a conserved tyrosine. However these enzymes are structurally and mechanistically different.

16 Type I A type I topoisomerase cuts one strand of a DNA double helix, relaxation occurs, and then the cut strand is reannealed. Cutting one strand allows the part of the molecule on one side of the cut to rotate around the uncut strand, thereby reducing stress from too much or too little twist in the helix. Such stress is introduced when the DNA strand is "supercoiled" or uncoiled to or from higher orders of coiling. Type I topoisomerases are subdivided into two subclasses: type IA topoisomerases, which share many structural and mechanistic features with the type II topoisomerases, and type IB topoisomerases, which utilize a controlled rotary mechanism. Examples of type IA topoisomerases include topo I and topo III. In the past, type IB topoisomerases were referred to as eukaryotic topo I, but IB topoisomerases are present in all three domains of life. Like type II topoisomerases, type IA topoisomerases form a covalent intermediate with the 5' end of DNA, whereas the IB topoisomerases form a covalent intermediate with the 3' end of DNA. Recently, a type IC topoisomerase has been identified, called topo V. While it is structurally unique from type IA and IB topoisomerases, it shares a similar mechanism with type IB topoisomerase.

17 Figure Action of E. coli type I topoisomerase (Topo I) The DNA-enzyme intermediate contains a covalent bond between the 5 -phosphoryl end of the nicked DNA and a tyrosine residue in the protein (inset). After the free 3 - hydroxyl end of the red cut strand passes under the uncut strand, it attacks the DNA-enzyme phosphoester bond, rejoining the DNA strand. During each round of nicking and resealing catalyzed by E. coli Topo I, one negative supercoil is removed. (The assignment of sign to supercoils is by convention with the helix stood on its end; in a negative supercoil the front strand falls from right to left as it passes over the back strand (as here); in a positive supercoil, the front strand falls from left to right.)

18 Type II A type II topoisomerase cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then reanneals the cut strands. This class is also split into two subclasses: type IIA and type IIB topoisomerases, which possess similar structure and mechanisms. Examples of type IIA topoisomerases include eukaryotic topo II, E. coli gyrase, and E. coli topo IV. Examples of type IIB topoisomerase include topo VI. Type II topisomerases utilize ATP hydrolysis.

19 (a) Introduction of negative supercoils. The initial folding introduces no stable change, but the subsequent activity of gyrase produces a stable structure with two negative supercoils. Eukaryotic Topo II enzymes cannot introduce supercoils but can remove negative supercoils from DNA. (b) Catenation and decatenation of two different DNA duplexes. Both prokaryotic and eukaryotic Topo II enzymes can catalyze this reaction.

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21 Topoisomerase IA IB IIA IIB Metal Dependence ATP Dependence Single- or Double- Stranded cleavage? Cleavage Polarity Yes No Yes Yes No No Yes Yes SS SS DS DS 5' 3' 5' 5' Change in L ±1 ±N ±2 ±2

22 Functions of topoisomerases Accessing DNA Removing DNA Supercoils Strand Breakage during Recombination Chromosome Condensation Disentangling Intertwined DNA Topoisomerases as Drug Targets

23 Upcoming Lecture Meselson-Stahl experiment Variations in semiconservative theme

24 Assignments Write about different proposed mechanisms for DNA replication. [2.5] Describe in detail about different classes and sub classes of topoisomerases. [7.5] Explain about linking number, twist of DNA and Writhe. [2.5] Write difference between Type I and II Topoisomerases. [2.5] Write about the topological problem during DNA replication and its solution. [2.5] Write about the topological problems arising during replication of linear and circular DNA. [2.5] Write about positive and negative supercoiling [2.5] Hypothesize about the possible mechanism beside topoisomerases for solving the topological problem. [1] Deadline : 4 th April

25 References Brown TA. Genomes. 2nd edition. Oxford: Wiley- Liss; Chapter 13, Genome Replication. Available from: Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; Section 12.3, The Role of Topoisomerases in DNA Replication. Available from: