6. DNA, Genomes, ChromosomesSunday, January 26, 201411:07 PM Exam 2 Page 1In Eukaryotes: most of the DNA is in the nucleus. Exam 2 Page 2In Eukaryotes: most of the DNA is in the nucleus.The number and size of chromosomes vary widely among different organisms.•Bacterial chromosomes are single and circular.•Eukaryotic chromosomes are linear and distributed between multiple chromosomes.•46 chromosomes○23 maternal and 23 paternal○22 autosome pairs○1 sex chromosome pair ○Matching pairs are called homologous chromosomes○Humans •Eukaryotic DNA is packaged into chromosomes. Exam 2 Page 3Exam 2 Page 4Exam 2 Page 5The human genome is 3 meters long.DNA in the nucleus is packed with proteins in a complex called chromatin which look like beads on a string under a microscope.The "beads on a string" condense themselves into a chromosome. Histone H1 binds the linker DNA and contributes to the condensation of the beads on a string.7. Chromatin & DNA SynthesisThursday, January 30, 20143:53 PM Exam 2 Page 6Chromatin is equal parts DNA and protein in complex with DNA.Histones are major DNA packaging proteins. They are lysine and arginine rich.Core histones are histone H2A, H2B, H3, H4Histone octamer and 147 base pairs of DNA that wrap the octamer50 bp form a linker between adjacent nucleosomes (the string)Strong electrostatic attraction causes DNA to associate with histonesNucleosome - "The Bead" Exam 2 Page 7Composition of the nucleosome linked togetherHeterochromatin is highly condensed and is associated with gene silencing. Euchromatin is less condensed and is associated with gene expression. Exam 2 Page 8Relationship between higher order structure and gene expression.Covalent modifications to histone "tails" regulate chromatin structure and gene expression. Exam 2 Page 9Acetylation and methylation change the chemistry of lysine side chains. Histone code readers bind particular combinations of signals and attract additional protein complexes that regulate access to the DNA Exam 2 Page 10DNA goes through semi conservative synthesis: One strand templates to make two new strands. Individual nucleotides are linked together1.Two riboses are attached by a phosphate that bridge the 3' and 5' carbons. Called a phosphodiester linkage (bond)2.DNA synthesis is in the 5' to the 3' direction. Exam 2 Page 11DNA polymerase catalyzes the synthesis of DNAIncorporation of the wrong nucleotide is rare but significant: 1 mistake/100,000 nucleotides.The 3' to 5' exonuclease proofreading activity by DNA polymerase increases fidelity. Exam 2 Page 12Exam 2 Page 13Replication forks are regions of active replication.Replication of the leading and lagging strands of DNA is asymmetric.The replication machinery for both strands is organized as a large complex which coordinates synthesis of each strand.DNA polymerase requires a primer to begin synthesis; it can only add nucleotides to the 3' end of a polynucleotide strand.Lagging strand synthesis requires multiple primers.DNA primase serves to generate short RNA primers that provide the 3' OH for DNA polymerase to start synthesis. Each Okazaki fragment begins synthesis with an RNA primer8. DNA replication: mechanismTuesday, February 04, 20149:19 PM Exam 2 Page 14Connecting the Okazaki fragments involves degrading the RNA primer and ligating the nick.RNase H degrades RNA in the heteroduplexed region.DNA polymerase fills the gap.DNA ligase ligates the nick.DNA synthesis occurs on a single stranded template: helicase unwinds DNA at the replication fork and SSBP keeps it single stranded. Exam 2 Page 15Single strand DNA binding protein prevents hairpins from forming in single stranded regions. The sliding clamp protein regulates the association of DNA polymerases with the template and keeps it on the template. Exam 2 Page 16Exam 2 Page 17DNA polymerase movement and unwinding of the double stranded helix produces stress ahead of the replication fork. Exam 2 Page 18Topo I introduces transient single strand breaks by an ATP-independent mechanism.1.Topoisomerases untangle DNA by introducing transient breaks in the DNA Exam 2 Page 19Topo II introduces transient double strand breaks by an ATP-dependent mechanism.2. Exam 2 Page 20DNA polymerase is highly accurate, but some errors go undetected during synthesis. Proofreading is not enough.In strand-directed mismatch repair, MutS and MutL complex scans the genome identifying errors. Exam 2 Page 21DNA synthesis begins at a replication origin, which is a specific DNA sequence. Bi-directional synthesis.Replication bubble forms and grows as replication progresses.Two replication forks move away from each other.In E. Coli:9. DNA replication in cellsTuesday, February 04, 201410:52 PM Exam 2 Page 22In E. Coli:The replication origin is AT-rich and associates with Initiator proteins.Initiator proteins recruit a DNA helicase that unwinds the DNA.DNA primase synthesizes an RNA primer which provides a 3' OH for DNA polymerase.Bacterial chromosomes are circular and have one origin.One origin is sufficient to replicate a bacterial chromosome which is a few million base pairs long. Exam 2 Page 23Eukaryotic chromosomes are hundreds of millions of base pairs long.Eukaryotes replicate DNA at multiple locations (use multiple origins of replication) to keep pace with the rate of cell proliferation.The firing of replication origins is controlled by the cell cycle to insure that DNA is replicated only once before cell division. DNA replicating occurs in the S phase. Exam 2 Page 24Origin Recognition Complex (ORC) serves as the initiator protein in eukaryotes.ORC is bound to the DNA throughout the cell cycle but is activated by a kinase during the transition to S phase.Once the origin has fired, ORC remains inactive until the cell divides. ORC recruits a helicases! Exam 2 Page 25Cell doubles the amount of histones also.Histone proteins are produced before and during DNA synthesis.Chromatin is formed right after the passage of DNA polymerase to make a fully formed chromosome.Replication of the chromosomes involves dissociating histones ahead of the fork and distributing them to both strands behind the replication fork using enzymes and proteins called histone chaperones. Newly synthesized histones fill in the gaps. Exam 2 Page 26Chromosome end replication problem:Because it requires a primer, DNA polymerase alone cannot complete synthesis of the tip of the chromosome. Exam 2 Page 27Telomerase is an RNA template directed DNA polymerase that