Chapter 3 DNA Replication

(1)

台大農藝系遺傳學 601 20000 Chapter 3 slide 1

CHAPTER 3 DNA Replication

Peter J. Russell

edited by Yue-Wen Wang Ph. D. Dept. of Agronomy, NTU

(2)

Semiconservative DNA Replication

• _{1. Watson and Crick DNA model implies a}

mechanism for replication:

• a. Unwind the DNA molecule.

• _{b. Separate the two strands.}

(3)

• 2 .Three possible models were proposed for DNA

replication:

• _{a. Conservative model proposed both strands of one copy would}

be entirely old DNA, while the other copy would have both

strands of new DNA.

• b. Dispersive model was that dsDNA might fragment, replicate

dsDNA, and then reassemble, creating a mosaic of old and new

dsDNA regions in each new chromosome.

• c. Semiconservative model is that DNA strands separate, and a

complementary strand is synthesized for each, so that sibling

chromatids have one old and one new strand. This model was the

winner in the Meselson and Stahl experiment.

(4)

(5)

The Meselson-Stahl Experiment

• _{Animation: The Meselson-Stahl Experiment}

• _{1. Meselson and Stahl (1958) grew E. coli in a heavy}

(not radioactive) isotope of nitrogen,

15

N in the form of

15

NH

₄

Cl. Because it is heavier, DNA containing

15

N is

more dense than DNA with normal

14

N, and so can be

separated by CsCl density gradient centrifugation (Box

3.1).

• _{2. Once the E. coli were labeled with heavy}

15

N, the

researchers shifted the cells to medium containing normal

14

N, and took samples at time points. DNA was extracted

from each sample and analyzed in CsCl density gradients

(Figure 3.2).

(6)

Fig. 3.2 The Meselson-Stahl experiment, which showed that DNA replicates semiconservatively

(7)

Box Fig. 3.1 Equilibrium centrifugation of DNA of different densities in a cesium chloride

(8)

台大農藝系遺傳學 601 20000 Chapter 3 slide 8 • 3. After one replication cycle in normal 14N medium, all DNA had

density intermediate between heavy and normal. After two replication cycles, there were two bands in the density gradient, one at the

intermediate position, and one at the position for DNA containing entirely 14N.

• 4. Results compared with the three proposed models:

• a. Does not fit conservative model, because after one generation there is a single intermediate band, rather than one with entirely 15N DNA

and another with entirely 14N DNA.

• b. The dispersive model predicted that a single band of DNA of intermediate density would be present in each generation, gradually becoming less dense as increasing amounts of 14N were incorporated

with each round of replication. Instead, Meselson and Stahl observed two bands of DNA, with the intermediate form decreasing over time. • c. The semiconservative model fits the data very well.

(9)

Semiconservative DNA Replication in

Eukaryotes

• _{1. To visualize DNA of}

eukaryotic chromosomes

replicating, CHO

(Chinese hamster ovary)

cells are grown in

5-bromodeoxyuridine

(BUdR), a base analog for

thymine. After two rounds

of replication, mitotic

chromosomes are stained

with fluorescent dye and

Giemsa stain (Figure 3.3).

(10)

• _{2. DNA containing T stains darkly, while DNA}

containing two BUdR strands stains lightly. Observed

that after one generation, both chromatids stain the same,

each with one BUdR strand and one T strand. After two

generations, they stain differently and are called harlequin

chromosomes, one light (both strands have BUdR) and

one dark (one strand has BUdR and other strand has T).

• 3. Showed that eukaryotes also use semiconservative

DNA replication.

(11)

Enzymes Involved in DNA Synthesis

• _{1. First isolation of an enzyme involved in DNA}

replication was in 1955. Arthur Kornberg won the

1959 Nobel Prize in Physiology or Medicine for

this work.

(12)

DNA Polymerase I

• _{1. Accomplished in vitro synthesis of E. coli DNA. His}

reaction mixture included:

• a. DNA fragments (template).

• b. Radioactively labeled dNTPs (dATP, dGTP, dTTP and

dCTP).

• c. E. coli lysate.

• 2. Enzyme originally called the Kornberg enzyme, now

known as DNA Polymerase I. Once isolated, could

characterize its activity, showing that the above

components are required, along with Mg

21

ions for

(13)

Roles of DNA Polymerases

• Animation: DNA Biosynthesis: How a New DNA Strand is Made

• 1. All DNA polymerases link dNTPs into DNA chains (Figure 3.4). Main features of the reaction:

• a. An incoming nucleotide is attached by its 5’-phosphate group to the 3’-OH of the growing DNA chain. Energy comes from the dNTP

releasing two phosphates. The DNA chain acts as a primer for the reaction.

• b. The incoming nucleotide is selected by its ability to hydrogen bond with the complementary base in the template strand. The process is fast and accurate.

• c. DNA polymerases synthesize only from 5’ to 3’.

• 2. Two additional DNA polymerases were later isolated, DNA Pol II in 1970 and DNA Pol III in 1971.

(14)

(15)

(16)

The properties of DNA polymerases

• 3. The properties of these enzymes are (Table 3.1):

• a. DNA polymerase I is a single peptide encoded by polA. There are about 400 molecules in an E. coli cell. Replicates DNA in the 5’ →3’ direction. Has 5’ →3’ exonuclease activity to remove nucleotides from 5’ end of DNA or from RNA primer.

• b. DNA polymerase II is a single peptide encoded by polB. Number of molecules per E. coli cell isn’t known for certain, but probably around 10–20. Role in the cell is unknown.

• c. DNA polymerase III has 10 polypeptide subunits encoded by 10 different genes. The catalytic core of the enzyme is composed of three subunits, α (encoded by the dnaE gene), ε (dnaQ) and θ (holE). There are 10–20 molecules of this enzyme in an E. coli cell. Replicates DNA in the 5’ →3’ direction.

• 4. All three E. coli DNA polymerases have 3’ →5’ exonuclease (proofreading) activity.

(17)

Molecular Model of DNA Replication

• _{1. Table 3.2 shows key genes and DNA}

(18)

Initiation o Molecular Model of DNA

Replication

• 1. Replication starts at origin of replication, with denaturation to

expose the bases and create a bi-directional replication bubble. E. coli has one origin, oriC, which has a minimal sequence of about 245 bp required for initiation.

• 2. Events in initiating DNA synthesis, derived from in vitro studies (Figure 3.5):

• a. Gyrase (a type of topoisomerase) relaxes supercoils in the region. • b. Initiator proteins attach.

• c. DNA helicase (from dnaB) binds initiator proteins on the DNA, and denatures the region using ATP as an energy source.

• d. DNA primase (from dnaG) binds helicase to form a primosome, which synthesizes a short (11 6 1 nt) RNA primer. Primers begin with two purines, typically AG.

(19)

Fig. 3.5 Model for the formation of a replication bubble at a replication origin in

(20)

Semidiscontinuous DNA Replication

(21)

Fig. 3.6a, b Model for the events occurring around a single replication fork of the

(22)

Fig. 3.6c-e Model for the events occurring around a single replication fork of the

(23)

• _{1. When DNA denatures at the oriC, replication forks are}

formed. DNA replication is usually bi-directional, but will

consider events at just one replication fork (Figure 3.6):

• a. Single-strand DNA-binding proteins (SSBs) bind the ssDNA formed by helicase, preventing reannealing.

• _{b. Primase synthesizes a primer on each template strand.}

• c. DNA polymerase III adds nucleotides to the 3’ end of the

primer, synthesizing a new strand complementary to the template, and displacing the SSBs. DNA is made in opposite directions on the two template strands.

• _{d. New strand made 5’ → 3’ in same direction as movement of}

the replication fork is leading strand, while new strand made in opposite direction is lagging strand. Leading strand needs only one primer, while lagging needs a series of primers.

(24)

• 2. Helicase denaturing DNA causes tighter winding in other

parts of the circular chromosome. Gyrase relieves this

tension.

• _{3. Leading strand is synthesized continuously, while lagging}

strand is synthesized discontinuously, in the form of

Okazaki fragments. DNA replication is therefore

semidiscontinuous.

• _{4. Each fragment requires a primer to begin, and is extended}

by DNA polymerase III.

• 5. Okazaki data show that these fragments are gradually

joined together to make a full-length dsDNA chromosome.

DNA polymerase I uses the 3’-OH of the adjacent DNA

fragment as a primer, and simultaneously removes the RNA

primer while resynthesizing the primer region in the form of

DNA. The nick remaining between the two fragments is

(25)

Fig. 3.7 Action of DNA ligase in sealing the gap between adjacent DNA fragments to

(26)

• _{6. Key proteins are associated to form a replisome.}

Template DNA probably bends to allow synthesis

of both leading and lagging strands at the

replication fork (Fig. 3.8)

• _{7. Early stages of bidirectional replication are}

(27)

Fig. 3.8 Model for the “replication machine,” or replisome, the complex of key

(28)

(29)

Fig. 3.9 Diagram of the formation at a replication origin sequence of two replication

(30)

(31)

Replication of circular DNA and the

supercoiling problem

• _{1. Some circular chromosomes (e.g., E. coli) are}

circular throughout replication, creating a

theta-like (θ) shape (Fig. 3.10). As the strands separate

on one side of the circle, positive supercoils form

elsewhere in the molecule. Replication fork

moves about 500 nt/ second, so at 10 bp/turn,

replication fork rotates at 3,000 rpm.

• _{2. Topoisomerases relieve the supercoils,}

allowing the DNA strands to continue separating

as the replication forks advance. (Fig. 3.11)

(32)

Fig. 3.11 Diagram showing the unreplicated, supercoiled parent strands and the

(33)

Rolling Circle Replication

• _{1. Another model for replication is rolling circle, which is}

used by several bacteriophages, including ΦX174 (after a

complement is made for the genomic ssDNA) and λ (after

circularization by base pairing between the “sticky”

ssDNA cos ends)

• _{2. Rolling circle replication begins with a nick}

(single-stranded break) at the origin of replication. The 5’ end is

displaced from the strand, and the 3’ end acts as a primer

for DNA polymerase III, which synthesizes a continuous

strand using the intact DNA molecule as a template.

• 3. The 5’ end continues to be displaced as the circle

“rolls”, and is protected by SSBs until discontinuous

DNA synthesis makes it a dsDNA again.

(34)

Fig. 3.12 The replication process of double-stranded circular DNA molecules through

(35)

• _{4. A DNA molecule many genomes in length can be made}

by rolling circle replication. During viral assembly it is

cut into individual viral chromosomes and packaged into

phage head.

• 5. Bacteriophage λ, regardless of whether entering the

lytic or lysogenic pathway, circularizes its chromosome

immediately after infection.

• _{a. In a lysogenic infection, the circular DNA integrated into a}

specific site in the E. coli chromosome by a crossover event.

• b. In a lytic infection, rolling circle replication produces a long concatamer of λ DNA, and the a viral endonuclease (product of the ter gene) recognize the cos sites and makes the staggered cuts that used to assemble new virus particles.

(36)

(37)

Fig. 3.14  chromosome structure varies at stages of lytic infection of E.

(38)

DNA Replication in Eukaryotes

• _{1. DNA replication is very similar in both}

prokaryotes and eukaryotes, except that

(39)

DNA Replication and the Cell Cycle

• 1. Each eukaryotic chromosome must be copied, so both DNA and histones must be doubled in each cell cycle. Chromosome duplication occurs during the S phase of the cell cycle, and segregation of progeny chromosomes during the M phase.

• 2. Details of cell cycle control are beyond the scope of this discussion. Yeasts, in which chromosomal replication is well studied, serve as a eukaryotic model organism.

• 3. Many genes are involved in the system of checks and balances that controls the cell cycle (Figure 3.15). Some major checkpoints in the system:

• a. To move from G1 into S phase, cells must be large enough, and the environment favorable. This checkpoint is called START in yeasts and G1 checkpoint in mammalian cells.

• b. The G2 checkpoint controls entry into M phase, assessing whether DNA has been duplicated, the cell is large enough and the environment is favorable.

• c. Attachment of chromosomes to the mitotic spindle during M phase functions as another checkpoint, triggering separation of the

(40)

台大農藝系遺傳學 601 20000 Chapter 3 slide 40 • 4. Checkpoints use cyclin proteins and cyclin-dependent kinases

(Cdks). Yeasts use a single Cdk for both the G1 and G2 checkpoints, while mammals use at least two Cdks for each checkpoint. In the yeast system, for example:

• a. At the START (G1) checkpoint, one or more G1 cyclins bind and activate the Cdk. The activated Cdk phosphorylates proteins needed to initiate S phase. Cyclin levels then decrease by proteolysis.

• b. At the G2 checkpoint, mitotic cyclin(s) bind to Cdk, forming the M-phase promoting factor (MPF). Dephosphorylation of MPF by other enzymes activates it, and it then catalyzes phosphorylation of proteins that move the cell into M phase. Mitotic cyclin is degraded after

metaphase, causing MPF inactivation so that mitosis can be completed. • 5. Cell cycle control is very complex, and will be discussed more fully

(41)

Fig. 3.15 Some of the molecular events that control progression through the cell cycle

(42)

Eukaryotic Replication Enzymes

• 1. Enzymes of eukaryotic DNA replication aren’t as well characterized as their prokaryotic counterparts. The replication process is similar in both groups—DNA denatures, replication is semiconservative and semidiscontinuous and primers are required.

• 2. Five DNA polymerases are known in mammalian cells:

• a. α (alpha) is nuclear, uses RNA primers, is involved in nuclear DNA

replication and has not been shown to proofread.

• b. β (beta) is nuclear, serves in DNA repair and does not show

proofreading activity.

• c. δ (delta) is nuclear, uses RNA primers, is involved in nuclear DNA

replication and is capable of proofreading.

• d. γ (gamma) is mitochondrial, replicating the mitochondrial DNA using

RNA primers and proofreading.

• e. ε (epsilon) is nuclear, has proofreading activity and may be used for

(43)

Replicons

• _{1. Eukaryotic chromosomes generally contain much more}

DNA than those of prokaryotes, and their replication

forks move much more slowly. If they were like typical

prokaryotes, with only one origin of replication per

chromosome, DNA replication would take a very long

time.

• 2. Instead, eukaryotic chromosomes contain multiple

origins, at which DNA denatures and replication then

proceeds bidirectionally until an adjacent replication fork

is encountered. The DNA replicated from a single origin

is called a replicon, or replication unit (Figure 3.16).

(44)

(45)

• _{3. Replicon size is smaller in eukaryotes than}

prokaryotes, and each chromosome contains many

replicons. Number and size of replicons may vary

with cell type.

• _{4. Not all origins within a genome initiate DNA}

synthesis simultaneously. Cell-specific patterns of

origin activation are observed, so that

chromosomal regions are replicated in a

predictable order in each cell cycle.

(46)

Fig. 3.17 Temporal ordering of DNA replication initiation events in replication units

(47)

Origins of Replication

• 1. Eukaryotic origins are generally not well characterized; those of the yeast Saccharomyces cerevisiae are among the best understood.

• 2. Chromosomal DNA fragments that are able to replicate

autonomously when introduced into yeast as extracellular, circular DNA are known as ARSs (autonomously replicating sequences).

• 3. Some ARSs appear to be yeast origins of replication, but others probably are not.

• _{4. Four sequence elements have been identified in ARSs. The A}

element is essential to the ARS, and combines with B1 and/or B2

elements to form the core of the ARS to which the multiprotein origin recognition complex (ORC) binds. The B3 element binds a protein called Abf1 (ARS-binding factor) to enhance replication.

(48)

Replicating the Ends of Chromosomes

• _{1. When the ends of chromosomes are replicated}

and the primers are removed from the 5’ ends,

there is no adjacent DNA strand to serve as a

primer, and so a single-stranded region is left at

the 5’ end of the new strand. If the gap is not

addressed, chromosomes would become shorter

with each round of replication (Figure 3.18).

(49)

Fig. 3.18 The problem of replicating completely a linear chromosome in eukaryotes

(50)

台大農藝系遺傳學 601 20000 Chapter 3 slide 50 • _{2. Most eukaryotic chromosomes have short, species-specific}

sequences tandemly repeated at their telomeres. Blackburn and Greider have shown that chromosome lengths are maintained by telomerase, which adds telomere repeats without using the cell’s regular replication machinery.

• 3. In the ciliate Tetrahymena, the telomere repeat sequence is 5’-TTGGGG-3’. Telomerase, an enzyme containing both protein and RNA, binds to the terminal telomere repeat when it is single-stranded, synthesizing a 3-nt sequence, TTG. The 3’ end of the telomerase RNA contains the sequence AAC, which binds the TTG positioning

telomerase to complete its synthesis of the TTGGGG telomere repeat. Additional rounds of telomerase activity lengthen the chromosome by adding telomere repeats.

• _{4. After telomerase adds telomere sequences, chromosomal replication}

proceeds in the usual way. Any shortening of the chromosome ends is compensated by the addition of the telomere repeats.

• 5. Telomere length may vary, but organisms and cell types have

characteristic telomere lengths. Mutants affecting telomere length have been identified, and data indicate that telomere length is genetically controlled. Shortening of telomeres eventually leads to cell death, and this may be a factor in the regulation of normal cell death.

(51)

(52)

Assembling New DNA into Nucleosomes

• 1. When eukaryotic DNA is replicated, it complexes with histones. This requires synthesis of histone proteins and assembly of new nucleosomes.

• 2. Transcription of histone genes is initiated near the end of G1 phase, and translation of histone proteins occurs throughout S phase.

• 3. Newly replicated DNA is organized into nucleosomes very quickly. DNA synthesis requires nucleosomes to disassemble so the DNA can be denatured, but only 200–300 bp will be nucleosome-free around the replication forks.

• 4. Data strongly suggest that new nucleosomes are composed of

entirely new histones, and existing nucleosomes are conserved. Old and new nucleosomes bind randomly to the sibling chromatids after

(53)

(54)