DNA Replication

• DNA replication- is a sequence of repeated condensation (dehydration synthesis) reactions linking nucleotide monomers into a DNA polymer.

  -it is the process that synthesizes DNA by copying existing DNA
• DNA replication proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation, and termination
• 3 types of DNA replication/ alt. hypotheses for DNA replication:

  1. Semiconservative rep.- parental DNA separate and each strand is a template, the daughter DNA has 1 old & 1 new strand.

  2. Conservative rep.- parental DNA is template for synthesis of new molecule.

  3. Dispersive rep.- daughter molecule old DNA interspersed w/ newly synthesized DNA.
• -DNA must be unwound to replicate

  -Topoisomerases catalyze changes in supercoiled state of DNA.

  -DNA rep. is very accurate since it allows 1x10^-8 mistakes/bases.
• DNA helicase- breaks the H bonds between A-T and C-G that created a Y-shape called replication fork.
• 5'-3' direction: leading strand (continuous rep)

  3'-5' direction: lagging strand (discontinuous rep by Okazaki fragment)
• In leading strand: RNA primer produce by primase binds at the end of the leading strand that acts as starting point for DNA synthesis.
• DNA polymerase: binds to leading strand and synthesizes new DNA by adding new complementary nucleotide in 5'-3' direction.
• In lagging strand: numerous RNA primers made by primase bind at various points along the strand. Chunks of DNA called Okazaki fragments added to the strand in a 5' to 3' direction.
• DNA ligase- seals up DNA into 2 continuous double strands.
• DNA Replication end result: 2 identical copies of DNA molecule that are semi-conservative.
• 8 Enzymes in Prokaryotic Replication

  1. Helicase: unwinds DNA strand forming y-shaped replication fork.

  2. Topoisomerase- relieves torsional that arises ahead from rep. fork when helicase unwinds DNA.

  3. Primase- creates RNA primers from DNA template serves as starting/priming point for DNA synthesis.

  4. DNA polymerase III- polymerizes by forming phosphodiester bond, continuous synthesis and lengthen the DNA strand in presence of RNA primer.
• 8 Enzymes in Prokaryotic Replication (CONT.)

  5. Single strand DNA-binding protein (SSB)- binds to single stranded DNA to stabilize it, so H bonding of DNA bases are oriented toward incoming nucleotide.

  6. Sliding clamp- protein encircles DNA strand and helps hold or stay the DNA polymerase to the strand throughout the replication cycle.

  7. DNA polymerase I- has nucleases to remove RNA primers and polymerizes the gap/space. in the strand.

  8. DNA ligase- catalyzes covalent bonding among base pairs and seals Okazaki fragments in the lagging strand. Sealing of sequences in the DNA strand.
• DNA is polymorphic and dynamic.

  -It can assume many conformation depends on:

  1. environmental conditions

  2. activity of the part involved

  3. need of the organism
• 3 forms of DNA: B-DNA, A-DNA and Z-DNA
• B-DNA: the canonical right handed DNA helix that is the most common form of DNA. The standard form conformation adopted by nearly all sequences within a genome.

  -10.5 bp per helical turn and distance between bases ( vertical rise) is 3.4 Å
• A-DNA: right-handed antiparallel helical duplex - with 11 base pairs per helical turn

  -base pairs are tilted to about 20° with respect to the helical axis

  -vertical rise between bp is 2.55 Å
• Z-DNA: left-handed, the most underwound form of the double-helix, has been mostly found in alternating purine-pyrimidine sequences (CG)n and (TG)n (results to zigzag backbone)

  -12 bp per helical turn, with bases shifted to the periphery of the helix

  -an average rise is 3.7 Å/bp

  -usually in locations near the site of transcription initiation
• Z-DNA Functions:

  -a sink for the torsional tension (superhelical tension) in negatively supercoiled DNA

  -helps to maintain the gene (close to it) in its activated, nucleosome-free state (nucleosomes do not bind to the very rigid Z- DNA form) -regulation of gene expression

  -Z-forming sequences accumulate near the transcription start site of genes in humans and other eukaryotes
• Why do different forms of DNA exist?

  activity-wise and the type of environment the DNA is exposed to
• GC H bonds: 3

  AT H bonds: 2
• Other forms of DNA:

  H-DNA, Holiday Junction, G-Quadruplex and i-Motif
• H-DNA: formed when a single DNA strand invades the major groove of a DNA duplex

  -triple-stranded helices are favored in negatively supercoiled DNA

  -interaction between strands involves the Hoogsteen edge of the Watson-Crick base pairs of the duplex to form base triplets.

  -formed primarily in mirror repeat sequences (AGAGGGnnnGGGAGA)
• HJ (Holiday Junction): composed of 4 DNA strands. Essential intermediates in doublestrand break repair

  -recombination, dependent DNA lesion repair, viral integration, restarting of stalled replication forks

  -proper segregation of homologous chromosomes during meiosis
• G-quadruplexes: 4 DNA strands assembled from guanine-rich sequences. Cyclic arrangement of 4 guanine into G-tetrads.

  -telomeric DNA repeats (3’- overhangs at chromosome ends)

  -centromeric sequences and in the immunoglobulin switch region
• I-motifs (Intercalated): composed of 4 strands, sequences rich in cytosine.

  -comprised of two parallel-stranded DNA duplexes held together in an antiparallel orientation by intercalated, C-C bp

  -stabilized by acidic conditions
• Eukaryotic DNA polymerases and their functions

  
• Eukaryotic DNA polymerases and their functions

  
• Exonucleases- work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain.

  Endonucleases- cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain.
• 1. 5’ to 3’ exonuclease- which is a dependent decapping protein

  2. 3’ to 5’ exonuclease- non independent protein

  3. poly(A)- specific 3’ to 5’ exonuclease
• Primer - provides a 3’ free -OH end to which DNA polymerase can attach the appropriate nucleotide. It is needed to initiate DNA synthesis by a DNA polymerase.
• Error Correction in DNA Replication:

  1. Replication Error: error occurs from replication , it can be:

  A. Mispairing- addition of nucleotide w/ an incorrect base

  B. Insertion/Deletion of nucleotide this affects transcription and translation.

  C. Strand slippage: strand slip during rep. causing shorten or elongate the new strand.

  2. DNA Damage: chemical damage, radiation, attack by water causing damage or loss to the base group of nucleotide.
• Repair Systems: to repair replication error/s, it can be:

  1. Excision Repair System:

  a. Base-pair excision repair

  b. Nucleotide excision repair

  c. Short-patch and long-patch excision repair

  2. Mismatch Repair: it marks the spot with a cut where the mismatch bp is and then uses an exonuclease to digest or "eat up" the DNA at the marker
• A. Short-patch excision repair - enzymes will recognize and remove "short patches" of DNA that are damaged and short patches of damage arise from bulky lesions such as thymine dimers

  B. Long-patch excision repair - involves a longer segment of DNA
• Types of RNA:

  A. Messenger RNA (mRNA) - carries information from DNA to ribosomes, the site of protein synthesis and is coding RNA

  B. transfer RNA (tRNA), ribosomal RNA (rRNA) - non-coding and key elements in the translation process, and various types of RNA regulators.

  C. Ribozymes - able to catalyze chemical reactions such as cut and join other RNA molecules and they also form peptide bonds between amino acids
• Gene Function:

  1. Gene-Enzyme Relationship- gene codes for enzyme (protein) that catalyzes chemical reactions in a metabolic pathway.

  2. One Gene-One Enzyme Hypothesis: each gene is responsible for directing the building of a single specific enzyme.

  3. Protein Structure: a. covalent (peptide, disulfide bonds) and b. non-covalent interactions (H bonding, ionic interactions, Van der Waals interactions, hydrophobic bonds)

  4. Collinearity of DNA and Proteins: The concept of collinearity as proven by protein being synthesized based on instructions from DNA.

  5.The Genetic Code and its Universality
• Genetic Code - the nucleotide base sequence on DNA which will be translated into sequence of amino acids of a protein

  -each amino acid is represented by a triplet of bases (codon), read in 5’ to 3’ direction
• Characteristics of the Genetic Code:

  1. It is a triplet code.

  2. It is non-overlapping.

  3. It is degenerate (or redundant) since many amino acids have more than one codon.

  4. It is almost universal.

  5. There are start and stop codons which serve as start and stop signals.

  6. There is a wobble in the anticodon, a single tRNA can recognize 3 different codons at most.
• stop codons: UAA, UAG, UGA

  start codon: AUG