DNA Replication Notes PDF

12/24/24 Google List Explain in detail the following: Structure of DNA Structure of RNA Photo 51 Oswald Avery Who is Erwin Chargaff (1950)? - important finding on base composition (by paper chromatography) What are Chargaff’s rules? Rule #1 : Base pairing rule i.e In DNA, percent guanine will match percent cytosine, and percent thymine will math percent adenine Rule #2: Composition of DNA varies between species. Example: Human genome (40% C/G and 60% A/T), and yeast genome (38% C/ G and 62% A/T) but the same in different tissues in the same organism. 1 12/24/24 2 12/24/24 RNA polymerase synthesizes RNA from DNA that is functionally for protein-coding (messenger RNA, mRNA) or non-coding (RNA genes). Because of these functions, RNA molecules are of following types: messenger RNA (mRNA) – It is the RNA that carries information from DNA to the ribosomes (site of protein synthesis) in the cell. The mRNA code sequences determine the amino acid sequence in the protein that is produced. ribosomal RNA (rRNA) – It incorporates into the ribosomes. transfer RNA (tRNA) – It is used to transfer specific amino acids to growing polypeptide chains at the ribosomal site of protein synthesis during translation. Other RNA: snRNA, miRNA, snoRNA, lncRNA, catalytic RNA (ribozymes) DNA vs RNA https://www.youtube.com/watch?v=6L3zO8t1lsE 3 12/24/24 https://www.kcl.ac.uk/the-story-behind-photograph-51  Concept of Protein vs DNA?  DNAas the transforming molecule or inheritance molecule. 4 12/24/24 5 12/24/24  Strand Separation  Proteins - Helix destabilizing proteins (HD proteins) & DNA helix- HD Protein Helicase unwinding proteins  DNA topoisomerases  Template DNA polymerase  RNA primer  Chain elongation Leading strand Lagging strand  Excision  Ligase Strand Separation  For replication of two individual strands, they must first separate in a small region since polymerases use only single stranded DNA as a template.  In prokaryotes – Replication begins at a single discrete site – the Origin of replication (Ori).  In eukaryotes – Replication begins at multiple sites along the DNA helix (for rapid replication of great length of eukaryotic DNA).  Formation of a ‘V’ (where active synthesis occur) when two strands unwind & separate. This region is called as ‘Replication Fork”.  Replication fork moves along the DNA as synthesis occurs.  Replication of double stranded DNA is bidirectional.  Replication fork moves in both directions away from the origin. 6 12/24/24 A special group of proteins is responsible for maintaining:  Separation of parental strands.  Unwinding the double helix ahead of advancing replication fork. Helix destabilizing proteins (HD proteins)  Also called Single stranded DNA binding proteins (SSB). ¡ bind cooperatively. ¡ bind non-enzymatically to single stranded DNA. ¡ are not enzymes. ¡ shift the equilibrium between double stranded DNA (ds DNA) and single stranded DNA (ss DNA) in the direction of ss DNA. ¡ keep the two strands of DNA separated in the area of replication origin, thus providing the necessary single stranded template. ¡ also protect the DNA from nucleases that cleave single stranded DNA. 7 12/24/24 DNA helix-unwinding proteins  Also called DNA helicases. ¡ possess enzymatic activity. ¡ bind to ss DNA near the replication fork & then move into the neighboring double stranded region, forcing the strands apart. ¡ require energy provided by ATP.  HD protein (SSB protein) HD Protein  DNA Helicase Helicase Leading strand DNA polymerase Lagging strand 8 12/24/24 Enzymes & protein: Helicase SSB protein DNA polymerase III Primase DNA polymerase I DNA ligase Type I DNA topoisomerases  Reversibly cut a single strand of the double helix.  Have both nuclease (strand cutting) & ligase (strand resealing) activities.  Do not require ATP  Store the energy from the phosphodiester bond they cleave, reusing the energy to reseal the strand.  By creating a transient ‘nick’, the DNA helix on either side of the nick is allowed to rotate the phosphodiester bond opposite the nick, thus relieving accumulated super-twists. 9 12/24/24 Type II DNA topoisomerases (DNA gyrase)  Bind tightly to both strands of DNA  Make transient breaks in both strands of DNA  Use ATP  Finally reseals the break.  Results negative super-twists that allow easier unwinding of DNA double helix.  Both parental strands serve as templates for DNA biosynthesis at the replication fork, producing two daughter molecules each of which contains two DNA strands in antiparallel orientation. DNA polymerases (for copying templates):  ‘Read’ the partial nucleotide sequence in the 3’ → 5’ direction.  Synthesize new DNA strand in 5’ → 3’ direction.  Therefore, the two newly synthesized nucleotide chains must grow in opposite directions, Ø one in the 5’ → 3’ direction towards the replication fork. Ø one in the 5’ → 3’ direction away from the replication fork.  This is accomplished by a slightly different mechanism on each strand (leading vs lagging theory). 10 12/24/24  The strand that is being copied in the direction of the advancing replication fork (Leading strand) is synthesized almost continuously. HD Protein Helicase  The strand that is being copied in the direction away from the replication fork is synthesized discontinuously with small fragments (‘Okazaki fragments’) being copied near the replication fork. These fragments eventually join to DNA polymerase become a single continuous strand (‘Lagging strand’). Leading strand Lagging strand  DNA polymerases can not initiate synthesis of a complimentary strand of DNA on a single stranded template.  They require a primer (short, double stranded region with free OH group on the 3’ end of short strand).  The OH group serves as the first acceptor of a nucleotide from DNA polymerase.  This free OH group is provided by short stretch of RNA. 11 12/24/24 3’ DNA template 5’ Primer Template New DNA  DNA chain elongation is catalyzed by DNA polymerase III.  Using 3’ OH group of RNA primer, DNA polymerase III begins to add nucleotides along the single stranded template which specifies the sequence of bases in the newly synthesized chain. HD Protein Helicase  The new strand: Ø grows in 5’→3’ direction. Ø is anti-parallel to the parental strand. The nucleotide building blocks are 5’ deoxyribonucleoside DNA polymerase  triphosphates.  All four deoxyribonucleoside triphosphates (dATP, dTTP, dCTP & dGTP) must be present. Leading strand Lagging strand  DNA chain growth can be blocked by certain nucleotide analogues (modified in the sugar portion). By blocking DNA replication, they slow the cell division. 12 12/24/24  Highly important for the survival of the organism that nucleotide sequence of DNA be replicated with as few errors as possible.  Misreading of template sequence may result in HD Protein Helicase deleterious mutations.  To ensure replication fidelity, DNA polymerase III has: 5’→3’ polymerase activity DNA polymerase ¡ ¡ 3’→5’ exonuclease activity (proof reading activity) As each nucleotide is added to the chain, DNA Leading strand Lagging strand  polymerase III checks to make sure the added nucleotide is correctly matched to its complimentary base on the template & edits its mistakes. Examples:  If the template base is ‘A’ & enzyme mistakenly inserts ‘C’ instead of ‘T’ HD Protein Helicase into the new chain, DNA polymerase III hydrolytically removes the misplaced ‘C’ & replaces it with the correct ‘T’. DNA polymerase  The 3’→5’ exonuclease activity Leading strand Lagging strand degrades only improperly base paired nucleotides from the end. 13 12/24/24  DNA polymerase III continues to synthesize DNA until it is blocked by a stretch of RNA primer. When this occurs, the RNA is excised & the gap filled by DNA HD Protein Helicase polymerase I.  DNA polymerase I has: ¡ 5’→3’ polymerase activity (for DNA synthesis) DNA polymerase ¡ 3’→5’ exonuclease activity (for proof reading the newly synthesized DNA) Leading strand Lagging strand ¡ 5’→3’ exonuclease activity (for hydrolytic removal of primers)  DNA polymerase I locates the space (nick) between the 3’ end of newly synthesized DNA & the 5’ end of the adjacent RNA primer.  Hydrolytically removes the RNA nucleotides HD Protein Helicase ‘ahead’ of itself moving in 5’→3’ direction.  Replaces the removed nucleotides with deoxyribonucleotides, synthesizing DNA in 5’→3’ direction. During synthesis it also “proof reads” DNA polymerase the new chain using 3’→5’ exonuclease activity.  This removal/ synthesis/ proofreading continues Leading strand Lagging strand until the RNA is totally degraded & the gap is filled with DNA. 14 12/24/24  The 5’→3’ exonuclease activity of DNA polymerase I differs from 3’→5’ exonuclease activity used by both DNA polymerase I & DNA polymerase III in many ways. HD Protein Helicase  5’→3’ exonuclease removes one nucleotide at a time from a properly base paired region of DNA. The removed nucleotides can be either ribonucleotides or deoxyribonucleotides. DNA polymerase  It can also remove mismatched ends in the 5’→3’ direction, removing 1─10 nucleotides at a time. Leading strand Lagging strand Template strand New DNA Completed daughter strand 15 12/24/24  The final phosphodiester linkage between the 5’ phosphate group on the DNA chain synthesized by DNA polymerase III & the 3’ OH group on the chain made by DNA polymerase I is catalyzed by DNA ligase.  The joining of these two stretches of DNA requires Template strand energy, which in humans is provided by the cleavage of ATP to AMP & PPi. New DNA Completed daughter strand In eukaryotes, there are at least 15 DNA polymerases, each with specific functions 1. DNA polymerase α (alpha) - lays down primers on the lagging strand (also known as RNA polymerase or DNA primase) 2. DNA polymerase Β (Beta) - editing and repair 3. DNA polymerase Υ (Upsilon) - unique to mitochondria 4. DNA polymerase δ (delta) - responsible for elongation of both leading and lagging strands 5. DNA polymerase ε (epsilon) - elongation of leading strand in some species 16 12/24/24  DNA study  https://www.youtube.com/watch?v=MeKRueznCr8 17

DNA Replication Notes PDF

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