1. Chapter 3 DNA Replication II: Detailed Mechanism

2. Initiation Initiation of DNA replication means primer synthesis. Different organisms use different mechanisms to make primers. Different phages infect E. coli using quite different primer synthesis strategies. Coliphages were convenient tools to probe DNA replication as they are so simple they must rely primarily on host proteins to replicate their DNAs. Already explained in detailed with diagram………………. See before chap

3. Elongation Once a primer is in place, real DNA synthesis can begin. An elegant method of coordinating the synthesis of lagging and leading strands keep the pol III holoenzyme engaged with the template. Replication can be highly processive and so very rapid. Direction of fork movement Direction of synthesis Of lagging strand Direction of synthesis of leading strand Protein complexes of the replication fork: DNA polymerase DNA primase DNA Helicase ssDNA binding protein Sliding Clamp Clamp Loader DNA Ligase DNA Topoisomerase

4. Speed of Replication The pol III holoenzyme synthesizes DNA at the rate of about 730 nt/sec in vitro. The rate in vivo is almost 1000 nt/sec. This enzyme is highly processive both in vitro and in vivo. Pol III core alone is a very poor polymerase, after assembling 10 nt it falls off the template. Takes about 1 minute to reassociate with the template and nascent DNA strand A protein called “sliding clamp”, the  -subunit confers processivity on holoenzyme (means complete Pol III) allows it to remain engaged with the template.

5. The Role of the  -Subunit Core plus the  -subunit can replicate DNA processively at about 1,000 nt/sec Dimer formed by  -subunit is ring-shaped Ring fits around DNA template Interacts with  -subunit of the core to tether the whole polymerase and template together Holoenzyme stays on its template with the  -clamp Eukaryotic processivity factor called PCNA forms a trimer, also forms a ring that encircles DNA and holds DNA polymerase on the template

6. Termination Termination of replication is straightforward for phage that produce long, linear concatemers. Concatemer grows until genome-sized piece is snipped off and packaged into phage head. Bacterial replication – 2 replication forks approach each other at the terminus region Contains 22-bp terminator sites that bind specific proteins (terminus utilization substance, TUS) Replicating forks enter terminus region and pause Leaves 2 daughter duplexes entangled Must separate or no cell division

7. Termination in Eukarytoes Unlike bacteria, eukaryotes have a problem filling the gaps left when RNA primers are removed at the end of DNA replication If primer on each strand is removed, there is no way to fill in the gaps DNA cannot be extended 3’  5’ direction No 3’-end is upstream If no resolution, DNA strands would get shorter with each replication.

8. 1.Two replication forks result in a theta-like (  ) structure. 2.As strands separate, positive supercoils form elsewhere in the molecule. 3.Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate. Replication of circular DNA in E. coli

9. 1.Common in several bacteriophages including. 2.Begins with a nick at the origin of replication. 3.5’ end of the molecule is displaced and acts as primer for DNA synthesis. 4.Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly). 5.During viral assembly the DNA is cut into individual viral chromosomes. Rolling circle model of DNA replication

10. The Eukaryotic Cell Cycle The stages of mitosis and cell division define the M phase. G 1 is typically the longest part of the cell cycle; G 1 is characterized by rapid growth and metabolic activity. Cells that are quiescent, that is, not growing and dividing (such as neurons), are said to be in G 0 phase. The S phase is the time of DNA synthesis. S is followed by G 2, a relatively short period of growth when the cell prepares for division.

11. Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges: Major checkpoints in the system 1.Cells must be large enough, and the environment favorable. 2.Cell will not enter the mitotic phase unless all the DNA has replicated. 3.Chromosomes also must be attached to the mitotic spindle for mitosis to complete. 4.Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks). DNA replication in eukaryotes:

12. The Cell Cycle ( In eukaryotes) Controls the Timing of DNA Replication: Progression through the cell cycle is regulated by checkpoints. Checkpoints depend on cyclins and cyclin-dependent kinases (CDKs). Initiation of replication depends on the origin recognition complex (ORC). DNA replication occurs only once per cell cycle. Initiation of DNA replication is divided into two steps: 1)Licensing of replication origins (late M or early G1) 2)The activation of replication at the origins during S phase by the action of Cdc7-Dbf4 and S-CDK (the S phase cyclin- dependent kinases)

13. Each eukaryotic chromosome is one linear DNA double helix Average ~10 8 base pairs long With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days. Solution ---> DNA replication initiates at many different sites simultaneously. Rates are cell specific!

14. What about the ends (or telomeres) of linear chromosomes? DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. this gap is not filled, chromosomes would become shorter each round of replication! Solution: 1.Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. 2.Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of of new repeats. 3.Compensates by lengthening the chromosome. 4.Absence or mutation of telomerase activity results in chromosome shortening and limited cell division.

15. Summary of DNA replication  DNA Replication is the process in which the DNA within a cell makes an exact copy of itself.  DNA replication is the process where an entire double-stranded DNA is copied to produce a second, identical DNA double helix.  Helicase unwinds the double helix starting at a replication bubble.  The two strands separate as the hydrogen bonds between base pairs are broken.  Two replication forks form and the DNA is unwound in opposite directions.  After helicase has completed unwinding the DNA strand, Single strand Binding Proteins (SSB) keep the two strands from re-annealing (coming back together).  Primase is an RNA polymerase that makes the RNA primer.  These primers “tell” the DNA polymerase where to start copying the DNA.  The DNA polymerase starts at the 3’ end of the RNA primer of the leading stand CONTINUOUSLY.  DNA is copied in 5’ to 3’ direction.  DNA polymerase copies the lagging strand DIS- continuously.

16.  The dis-continuous pieces of DNA copied on the lagging strand are known as Okazaki fragments.  Another DNA Polymerase removes the RNA primers and replaces them with DNA.  Finally the gaps in the sugar phosphate backbone are sealed by DNA ligase. There are now 2 identical double helices of DNA.

17. How Are RNA Genomes Replicated? Many viruses have genomes composed of RNA. DNA is an intermediate in the replication of RNA viruses. The viral RNA is as a template for DNA synthesis. The RNA-directed DNA polymerase is called reverse transcriptase. All RNA tumor viruses contain such an enzyme within their viral particle. RNA viruses that replicate their RNA via a DNA intermediate are termed retroviruses. The primer for reverse transcriptase is a specific tRNA molecule captured from the host cell.  Reverse transcriptase transcribes the RNA template into a complementary cDNA strand to form a DNA:RNA hybrid Reverse transcriptase has three enzyme activities: 1)RNA-directed DNA polymerase activity 2)RNase H activity (an exonuclease activity that degrades RNA chains in DNA:RNA hybrids) 3)DNA-directed DNA polymerase activity (which replicates the ssDNA remaining after RNase H degradation of the viral genome, yielding a DNA duplex) which directs the remainder of the viral infection process.

18. How Are RNA Genomes Replicated? HIV reverse transcriptase is of great clinical interest because it is the enzyme for AIDS virus replication. DNA synthesis by HIV reverse transcriptase is blocked by nucleotide analogs such as AZT (3'-azido-2',3'- dideoxythymidine) and 3TC (2',3'-dideoxy-3'-thiacytidine). HIV reverse transcriptase incorporates these analogs into growing DNA chains in place of dTMP (in the case of AZT) or dCMP (in the case of 3TC) Once incorporated, these analogs block further chain elongation because the lack a 3'-OH where the next incoming dNTP can be added. The high error rate of HIV reverse transcriptase means that the virus is ever changing, which makes it difficult to devise an effective vaccine.

19. DNA recombination  DNA recombination refers to the process that a DNA segment moves from one DNA molecule to another DNA molecule.  Genetic recombination rearranges genetic information, creating new associations.  Meselson and Weigle showed that recombination involves exchange of DNA segments The following three types are most commonly observed. Homologous recombination It occurs between two homologous DNA molecules, also called DNA crossover. Site-specific recombination It occurs at a specific DNA sequence which is present in both non-homologous DNA molecules that may have the recombination. Transpositional recombination A mobile element is inserted into a target DNA.  The homologous recombination often occurs during meiosis. Other types of recombination are not specifically related to cell division.

20.  The process underlying homologous recombination is termed general recombination.  General recombination requires the breakage and reunion of DNA strands.  In 1964, Robin Holliday proposed a model for homologous recombination.  Two homologous DNA duplexes first juxtapose so that their sequences are aligned – a process of chromosome pairing called synapsis.  Recombination starts with introduction of small nicks at homologous sites on the two chromosomes.  Duplexes partially unwind, and the free, single-stranded end of one duplex begins to base-pair with its nearly complementary single-stranded region along the intact strand in the other duplex.  This process is called strand invasion. Ligation follows, forming a Holliday junction. Homologous Recombination Proceeds According to the Holliday Model

21. The + and – signs label strands of like polarity. For example, assume that the two strands running 5' to 3' as read are labeled +; and the two strands running 3' to 5' as read left to right are labeled -. Only strands of like polarity exchange DNA during recombination. The Holliday Model for Homologous Recombination

22. The Enzymes of General Recombination include RecA, RecBCD, RuvA, RuvB, & RuvC In E. coli, the principal players in recombination are: The RecBCD enzyme complex, which initiates recombination. The RecBCD complex is composed of RecB, RecC, and RecD and has both helicase and nuclease activity RecBCD initiates recombination by attaching to the end of a DNA duplex and using its helicase function to unwind dsDNA As it unwinds DNA, SSB binds to the single strands RecBCD endonuclease activity cleaves ssDNA RecBCD directs binding of RecA to 3'-terminal strand A nucleoprotein filament is formed This nucleoprotein filament is capable of homologous pairing with a dsDNA and strand invasion

23.  The RecA protein, which binds single-stranded DNA, forming a nucleoprotein filament capable of strand invasion and homologous pairing.  The RuvA, RuvB, and RuvC proteins, which drive branch migration and process the Holliday junction into recombinant products.  The Holliday junction is processed into recombination products by RuvA, RuvB, and RuvC.  RuvA and RuvB work together as a junction-specific helicase complex that dissociates the RecA filament and catalyzes branch migration.  Eukaryotic homologs of these prokaryotic proteins have been identified, indicating that the fundamental process of recombination is conserved across all organisms.

24. Transposons are DNA Sequences That Can Move from Place to Place in the Genome Barbara McClintock first proposed (in 1950) that activator genes could cause mutations in other genes. McClintock’s research showed (surprisingly) that activator genes could move about the genome. Her “jumping genes” model was viewed with skepticism at first, but molecular biologists verified her model in the 1970s, and in 1983, she was finally awarded the Nobel Prize in Physiology or Medicine for this remarkable discovery. McClintock’s jumping genes are now designated as mobile elements, transposable elements, or simply transposons

25. Transposons are DNA Sequences That Can Move from Place to Place in the Genome The typical transposon has inverted nucleotide-sequence repeats at its termini, represented here as the 12- bp sequence ACGTACGTACGT. (a) It acts as a target sequence by creating a staggered cut (b) whose protruding ends are ligated to the transposon (c). Gaps are filled in and ligated (d). Transposon insertion thus generates directed repeats of the target site in the host DNA.

26. Can DNA Be Repaired? A fundamental difference from RNA, protein or lipid All the others can be replaced, but DNA must be preserved. Cells require a means for repair of missing, altered or incorrect bases, bulges due to insertion or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links The human genome has about 150 genes associated with DNA repair. DNA repair systems include: direct reversal damage repair, single-strand damage repair, double strand break repair, and translesion DNA synthesis.

27. Can DNA Be Repaired? Chemical reactions that reverse the damage, returning DNA to its proper state, are direct reversal repair systems. Single-strand damage repair relies on the intact complementary strand to guide repair Systems repairing single-strand breaks include: Mismatch repair (MMR) Base excision repair (BER) Nucleotide excision repair (NER) Double-strand breaks (DSBs) are a particular threat to genome stability, because lost sequence information cannot be recovered from the same DNA