An-Najah National University Genetics: Analysis and Principles Robert J. Brooker CHAPTER 11 Lecture 6 DNA REPLICATION Dr. Heba Al-Fares

INTRODUCTION DNA replication is the process by which the genetic material is copied – The original DNA strands are used as templates for the synthesis of new strands It occurs very quickly, very accurately and at the appropriate time in the life of the cell – This chapter examines how!

DNA replication relies on the complementarity of DNA strands – The AT/GC rule or Chargaff’s rule The process can be summarized as such – The two DNA strands come apart – Each serves as a template strand for the synthesis of new strands – The two newly-made strands = daughter strands – The two original ones = parental strands 11.1 STRUCTURAL OVERVIEW OF DNA REPLICATION

Figure 11.1 Identical base sequences

In the late 1950s, three different mechanisms were proposed for the replication of DNA – Conservative model Both parental strands stay together after DNA replication – Semiconservative model The double-stranded DNA contains one parental and one daughter strand following replication – Dispersive model Parental and daughter DNA are interspersed in both strands following replication Experiment 11A: Which Model of DNA Replication is Correct?

Figure 11.2

In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models – They found a way to experimentally distinguish between daughter and parental strands Their experiment can be summarized as such – Grow E. coli in the presence of 15 N (a heavy isotope of Nitrogen) for many generations The population of cells had heavy-labeled DNA – Switch E. coli to medium containing only 14 N (a light isotope of Nitrogen) – Collect sample of cells after various times – Analyze the density of the DNA by centrifugation using a cesium chloride CsCl gradient

If both DNA strands contained 14N, the DNA would have a light density and sediment near the top of the tube. If one strand contained 14N and the other strand contained 15N, the DNA would be half-heavy and have an intermediate density. Finally, if both strands contained 15N, the DNA would be heavy and would sediment closer to the bottom of the centrifuge tube.

Figure 11.4 presents an overview of the process of bacterial chromosome replication – DNA synthesis begins at a site termed the origin of replication Each bacterial chromosome has only one – Synthesis of DNA proceeds bidirectionally around the bacterial chromosome – The replication forks eventually meet at the opposite side of the bacterial chromosome This ends replication 11.2 BACTERIAL DNA REPLICATION

Figure 11.4

The origin of replication in E. coli is termed oriC origin of Chromosomal replication Three types of DNA sequences in oriC are functionally significant AT-rich region DnaA boxes GATC methylation sites Refer to Figure 11.5 Initiation of Replication

DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them This generates positive supercoiling ahead of each replication fork DNA gyrase (a topoisomerase enzyme) travels ahead of the helicase and alleviates these supercoils Single-strand binding proteins bind to the separated DNA strands to keep them apart Then short (10 to 12 nucleotides) RNA primers are synthesized by DNA primase These short RNA strands start, or prime, DNA synthesis They are later removed and replaced with DNA

Figure 11.7 Breaks the hydrogen bonds between the two strands Alleviates supercoiling Keep the parental strands apart Synthesizes an RNA primer

DNA polymerases are the enzymes that catalyze the attachment of nucleotides to make new DNA In E. coli there are five proteins with polymerase activity DNA pol I, II, III, IV and V DNA pol I and III Normal replication DNA pol II, IV and V DNA repair and replication of damaged DNA DNA Polymerases

DNA pol I Composed of a single polypeptide Removes the RNA primers and replaces them with DNA DNA pol III Composed of 10 different subunits (Table 11.2) The  subunit synthesizes DNA The other 9 fulfill other functions The complex of all 10 is referred to as the DNA pol III holoenzyme DNA polIII can attach the nucleotides in 5' to 3' direction as it slides toward the opening of the fork (at a rate of 750 nucleotides per second). DNA Polymerases

Unusual features of DNA polymerase function Figure 11.9 Problem is overcome by the RNA primers synthesized by primase DNA polymerases cannot initiate DNA synthesis DNA polymerases can attach nucleotides only in the 5’ to 3’ direction Problem is overcome by synthesizing the 3’ to 5’ strands in small fragments

The two new daughter strands are synthesized in different ways Leading strand One RNA primer is made at the origin DNA pol III attaches nucleotides in a 5’ to 3’ direction as it slides toward the opening of the replication fork Lagging strand Synthesis is also in the 5’ to 3’ direction However it occurs away from the replication fork Many RNA primers are required DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each) These are termed Okazaki fragments after their discoverers

DNA pol I removes the RNA primers and fills the resulting gap with DNA It uses its 5’ to 3’ exonuclease activity to digest the RNA and its 5’ to 3’ polymerase activity to replace it with DNA After the gap is filled a covalent bond is still missing DNA ligase catalyzes a phosphodiester bond Thereby connecting the DNA fragments

Figure 11.7 Breaks the hydrogen bonds between the two strands Alleviates supercoiling Keep the parental strands apart Synthesizes an RNA primer Synthesizes daughter DNA strands III Covalently links DNA fragments together

20 Termination of replication in Bacteria Replication is terminated when the two replication forks meet at the terminus sequences. On the side of the chromosome opposite to the oriC are two termination sequences, T1 and T2 (called ter sequences). A protein known as the termination utilization substance (Tus) binds to the ter sequences and stops the movement of the replication forks. DNA replication ends when oppositely advancing forks meet at T1 or at T2.

21 Finally, DNA ligase covalently links the two daughter strands forming two circular double-stranded DNA molecules. Topoisomerases introduce a temporary break into the DNA strands then rejoin them after the strands have been unlocked. Although mistakes can occur during DNA replication, they are very rare. In the case of DNA synthesis by pol III, only one mistake per 100 million nucleotides is made.

22 The reasons that there are very few mistakes are: 1.Hydrogen bonding between matched base pairs is more stable than that between mismatched pairs. 2.DNA polymerase is unlikely to catalyze bond formation between adjacent nucleotides if a mismatched base pair is formed. 3.DNA polymerase can identify a mismatched pair and remove it by exonuclease cleavage (this ability to remove mismatched pairs is called the proofreading function of DNA polymerase).

Eukaryotic DNA replication is not as well understood as bacterial replication – The two processes do have extensive similarities, – Nevertheless, DNA replication in eukaryotes is more complex due to large linear chromosomes, tight packaging within nucleosomes, and more complicated cell cycle regulation Eukaryotes have long linear chromosomes – They therefore require multiple origins of replication To ensure that the DNA can be replicated in a reasonable time 11.3 EUKARYOTIC DNA REPLICATION

Figure Bidrectional DNA synthesis Replication forks will merge

 Initiation of replication in Eukaryotes occurs at multiple origins of replication.  DNA replication proceeds bidirectionally from many origins of replication. The multiple replication forks eventually meet with each other to complete the replication process.  In the yeast, several origins have been identified and sequenced. They have been named ARS elements (Autonomously Replicating Sequence).  ARS elements are bp in length and have higher percentage of A and T than the rest of the DNA.  It appears to be found in all eukaryotes Multiple Origins of Replication

Origin recognition complex (ORC) A six-subunit complex that acts as the initiator of eukaryotic DNA replication It appears to be found in all eukaryotes Requires ATP to bind ARS elements Single-stranded DNA stimulates ORC to hydrolyze ATP Multiple Origins of Replication

Mammalian cells contain well over a dozen different DNA polymerases, four of them have the primary function of replicating DNA: alpha (  ), delta (  ), epsilon (  ) and gamma (  ) ,  and    Nuclear DNA   Mitochondrial DNA Eukaryotes Contain Several Different DNA Polymerases

DNA pol  is the only polymerase to associate with primase. The DNA pol  /primase complex synthesizes a short RNA-DNA hybrid 10 RNA nucleotides followed by 20 to 30 DNA nucleotides This short RNA-DNA strand is then used by DNA pol δ or ε for elongation of the leading and lagging strands. The exchange of DNA Pol α for δ or ε is called a polymerase switch and only occurs after the RNA- DNA primer has been made Refer to Figure 11.21

Figure 11.21

DNA polymerases also play a role in DNA repair DNA pol  is not involved in DNA replication It plays a role in base-excision repair Removal of incorrect bases from damaged DNA Recently, more DNA polymerases have been identified Lesion-replicating polymerases Involved in the replication of damaged DNA They can synthesize a complementary strand over the abnormal region

Replication doubles the amount of DNA Therefore the cell must synthesize more histones to accommodate this increase Synthesis of histones occurs during the S phase Histones assemble into octamer structures They associate with the newly made DNA very near the replication fork Thus following DNA replication, each daughter strand has a mixture of “old” and “new” histones Refer to Figure Nucleosomes and DNA Replication

Figure Newly made histone proteins

33 Telomeres and DNA Replication Linear eukaryotic chromosomes contain telomeres at both ends. The term telomere refers to the telomeric sequences within the DNA and the special proteins that are bound to these sequences. These consist of repetitive tandem array of G and T and a 3`overhang region of 12 to 16 nucleotides. One reason why telomeric sequences are needed is because DNA polymerase is unable to replicate the 3`end of the DNA strands. Figure 11.23

DNA polymerases possess two unusual features 1. They synthesize DNA only in the 5’ to 3’ direction 2. They cannot initiate DNA synthesis These two features pose a problem at the 3’ end of linear chromosomes Figure 11.24

35 If this problem is not solved, the linear chromosome will become progressively shorter with each round of replication

36 Telomerase is an enzyme that adds telomere repeat sequences to the 3' end of DNA strands. By lengthening this strand, DNA polymerase is able to complete the synthesis of the "incomplete ends" of the opposite strand.

37 This figure shows how telomeres (red) that make up the ends of chromosomes, are formed. Telomere replication requires the action of telomerase (light green), which is a protein–RNA complex that carries an RNA strand (blue) that serves as a template for the formation of the G-rich telomere DNA sequence. After telomere replication, DNA synthesis can continue, and a new DNA strand formed

Figure Step 1 = Binding Step 3 = Translocation The binding- polymerization- translocation cycle can occurs many times This greatly lengthens one of the strands The complementary strand is made by primase, DNA polymerase and ligase RNA primer Step 2 = Polymerization