Chapter 11 DNA replication and recombination

• conservative
• semiconservative
• dispersive

original helix is conserved and two newly synthesized strands come together

each replicated DNA molecule consists of one "old"strand and one new strand

Parental strands are dispersed into two new double helices

used 15N and 14 N to demonstrate that DNA replication is semiconservative

• using broad bean vicia faba
• demonstrated that DNA replication is semiconservative in eukaryotes

• created when replication is occuring
• strands of helix are unwound
• Bidirectional; 2 replication forks at one origin site of replication

length of DNA that is replicated following one initiation event at a single origin

origin of replication in which bacteria have a single circular DNA and the DNA synthesis originates at this single point

catalyzes DNA synthesis and requires a DNA template and all four deoxyribonucleoside triphosphates (dNTPs)

• a nucleotide is added at the 3' end
• as the nucleotide is added the two terminal phosphates are cleaved off and a newly exposed 3'-OH group can participate in the addition of another nucleotide as DNA synthesis proceeds

enzyme responsible for the 5' to 3' polymerization essential in vivo

DNA polymerase III is a complex enzyme that is made up of 10 subunits

• complex formed at the replication fork
• consists of holoenzyme and some other proteins

• 1) unwinding of the helix
• 2) reducing increased coiling generated during unwinding
• 3) synthesis of a primer for initiation
• 4)discontinuous synthesis of the second strand
• 5) removal of the RNA primers
• 6) joining of the gap-filling DNA to the adjacent strand
• 7) proofreading

binds to the origin of replication and is responsible for the initial steps in unwinding the helix

further open and destabilizes the helix when binded

proteins which require the energy normally supplied by the hydrolysis of ATP to break hydrogen bonds and denature the double helix

stabilize the open conformation

produced from unwinding

• relieves supercoiling
• belongs to a larger group of enzymes referredt o as DNA topoisomerases
• Gyrase makes single or double stranded cuts to undo the twists and knots created during supercoiling which are then resealed

• synthesizes an RNA primer that provides the free 3-hydroxyl required by DNA polymerase III
• initiates DNA synthesis

removes primer and replaced with DNA

continuous DNA synthesis; the main template

• undergoes discontinuous DNA synthesis
• synthesized as Okazaki fragments, each wtih an RNA primer

joins Okazaki fragments after primers are removed by DNA polymerase I

prevents the core enzyme from falling off the template during DNA synthesis

• integral part of DNA replication
• All of the DNA polymerases have a 3' to 5' exonuclease activity that allows proofreading

DNA polymerase III, single-stranded binding proteins, DNA gyrase, DNA helicase, and RNA primers

a number of genes involved in DNA replication

• example of a conditional mutation
• may not be expressed at a particular permissive temperature but when mutant cells are grown at a restrictive temperature, the mutant phenotype is expressed and can be studied

• eukaryotes have more DNA than prokaryotes
• chromosomes are linear
• DNA is complexed with proteins
• eukaryotes contain multiple origins of replication to allow the genome to be replicated in a matter of mins to a few hours

• yeast genomes contain 250-400 origins
• these sequences contain an 11-bp consensus sequence flanked by other short sequences involved in efficient initiation

assembles at replication origins

• in early G1 phase of cell cycle
• replication origins are recognized by a six-protein complex
• tags the origin as the site of initiation

One

major forms of enzyme involved in initiation and elongation

term that reflects the length of DNA that is synthesized by an enzyme before it dissociates from the template

• possesses low processivity
• synthesis of RNA primers during initiation on the leading and lagging strands

once primer is in place, polymerase alpha and epsilon are replaced by polymerase delta for elongation

synthesizes lagging strand

synthesizes lagging strand

ends of linear chromosomes consists of long stretches of repeating sequences and preserve the integrity and stability of chromosomes

• directs synthesis of the telomere repeat sequence to fill the gap
• this is an enzyme consisting of a ribonucleoprotein with an RNA that serves as the template for the synthesis of its DNA complement (reverse transcription)

DNA is made form an RNA template

malignant cells maintain telomerase activity and are immortalized

• endonuclease nicking
• strand displacement and pairing with complement
• ligation
• branch migration
• duplex separation to generate the characteristic Holliday structure

genetic exchange at equivalent positions along two chromosomes with substantial DNA sequence homology

protein in E. coli promotes the exchange of reciprocal single-stranded DNA molecules and enhances hydrogen bond formation during strand displacement

characterized by nonreciprocal genetic exchange between two closely linked genes