1. Chapter 10 DNA Metabolism: Replication, Recombination, and Repair DNA polymerase & DNA Recombination

2. DNA Polymerase  This is the main enzyme that can synthesis a new strand on a template strand.  In case of Bacteria DNA polymerase I III II 5‘ 3‘ 5‘ 3‘ polymerase exonuclease ＋＋＋ ＋＋＋ ＋ －－ Replicase －－ ＋ MW (kDa) 103 90900 Numbers40010-20??? Bioactivity 10.0515 Gene pol C ＊ pol A pol B

3. Eukaryotic Cells Contain a Number of Different DNA Polymerases At least 19 different DNA polymerases have been found in eukaryotic cells so far. Multiple polymerases participate in leading- and lagging-strand synthesis, especially α, δ, and ε. α functions in initiation of nuclear DNA replication. Polymerase δ is the principal DNA polymerase in eukaryotic DNA replication. Through its association with PCNA (proliferating cell nuclear antigen), polymerase δ carries out highly processive DNA synthesis.

4. Eukaryotic Cells Contain a Number of Different DNA Polymerases

5. Why Are There So Many DNA Polymerases? Cells have different DNA polymerases for different purposes. Polymerases can be grouped in seven functional families, based on sequence homology. Family A includes polymerases involved in DNA repair in bacteria. Family B includes the eukaryotic polymerases involved in replication of chromosomal DNA Family C is that of the bacterial chromosomal DNA- replicating enzymes. Families X and Y act in DNA repair pathways RT designates retrovirus polymerases.

6. The Common Architecture of DNA Polymerases Despite sequence variation, the various DNA polymerases follow a common architectural pattern This common structure resembles a right hand, with distinct domains referred to as fingers, palm, and thumb The active site lies in a crevice within the palm domain The fingers act in deoxynucleotide recognition and binding The thumb is responsible for DNA binding

7. 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)

8. 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.

9. 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.

10. 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.

11.  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

12. 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

13. 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

14.  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.

15. 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

16. 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.