DNA Replication Abhi Nath MEDCH 570 [email protected] MedChem 570 ~ Medicinal Biochemistry: DNA Replication I. The Flow of Genetic Information The function of DNA is the storage of genetic information. DNA sequences known as genes specify the kinds of proteins that are made by cells. •DNA Replication refers to the synthesis of new DNA using the existing DNA as a template. •Transcription refers to the synthesis of RNA using a gene sequence (DNA) as a template. •Translation refers to the synthesis of a protein using an mRNA transcript as the template.The Flow of Genetic Information DNA Replication refers to the synthesis of new DNA using the existing DNA as a template. Nucleus Nucleus Cytosol Transcription refers to the synthesis of RNA using a gene sequence (DNA) as a template. Translation refers to the synthesis of a protein using an mRNA transcript as the template. Note that: Note that: • most human DNA is never transcribed. • not all RNA molecules are translated (e.g., rRNA, tRNA, others). • RNA is sometimes reverse transcribed back to DNA. •most human DNA is never transcribed •not all RNA molecules are translated (e.g., rRNA, tRNA, others) •RNA is sometimes reverse transcribed back to DNA. 1 The Structure of DNA is Fundamental to its Function The function of DNA is the storage of genetic information. DNA sequences known as genes specify the kinds of proteins that are made by cells. Complementary base-pairing ensures redundancy and allows for genetic information to be copied. Wikimedia Commons II. DNA Polymerases and DNA Replication The replication of DNA is effected by enzymes known as DNA polymerases. These enzymes catalyze the step-by-step addition of deoxynucleoside monophosphates to a DNA chain (the primer strand). DNA ReplicationMg2+ and DNA Polymerases DNAn + dNTP DNAn+1 + PPi The replication of DNA is effected This reaction constitutes a nucleophilic attack by enzymes known as DNA by the 3'-hydroxy group of the primer strand polymerases. on the α-phosphate of the incoming deoxynucleosideThese enzymes triphosphate. catalyze the step- by-step addition of deoxynucleoside monophosphates to a DNA chain This results in the formation of a covalent (the primerI Istrand. DNA). Polymerases and DNA Replication phosphodiester bond in a transesterificationThe reaction. replication of DNA is effected by enzymes known as DNA polymerases. These enzymes catalyze the step-by-step addition of deoxynucleoside monophosphates to a Note that polymerizationDNA proceeds chain (the in theprimer strand). 5"→3" direction. A driving force is the subsequent hydrolysisMg 2+ DNAn + dNTP DNAn+1 + PPi of PPi by inorganic pyrophosphatase. This reaction constitutes a nucleophilic attack by the 3'-hydroxy group of the primer strand on the α-phosphate of the incoming deoxynucleoside triphosphate. Requirements for DNA Replication by All DNA Polymerase Enzymes Template strandThis results in the formation of a covalent phosphodiester bond in a Primer strand with a free 3’ hydroxyl group transesterification reaction. 2’-deoxynucleoside 5’-triphosphates Mg2+ Note that polymerization proceeds in the 5"→3" direction. A driving force is the subsequent hydrolysis of PPi by inorganic pyrophosphatase. 2 Requirements for DNA Replication by All DNA Polymerase Enzymes Template strand Primer strand with a free 3’ hydroxyl group 2’-deoxynucleoside 5’-triphosphates 2+ Mg 2 II. DNA Polymerases and DNA Replication The replication of DNA is effected by enzymes known as DNA polymerases. These enzymes catalyze the step-by-step addition of deoxynucleoside monophosphates to a DNA chain (the primer strand). DNA ReplicationMg2+ and DNA Polymerases DNAn + dNTP DNAn+1 + PPi This reaction is a nucleophilic This reaction constitutes a nucleophilic attack attack by the 3'-hydroxy group of by the 3'-hydroxy group of the primer strand the primer strand on the α- on the -phosphate of the incoming phosphateα of the incoming deoxynucleosidedeoxynucleoside triphosphate. triphosphate. This results in the formation of a This results in the formation of a covalent covalent phosphodiester bond in a phosphodiester bond in a transesterification reaction. transesterification reaction. Note that polymerization proceeds in the 5’3’ direction. Note that polymerization proceeds in the 5"→3" direction.A driving force is the subsequent hydrolysis of PPi by inorganic A drivingpyrophosphatase force is the subsequent. hydrolysis of PPi by inorganic pyrophosphatase. Requirements for DNA Replication by All DNA Polymerase Enzymes Template strand Primer strand with a free 3’ hydroxyl group 2’-deoxynucleoside 5’-triphosphates 2+ Mg 2 II. DNA Polymerases and DNA Replication The replication of DNA is effected by enzymes known as DNA polymerases. These enzymes catalyze the step-by-step addition of deoxynucleoside monophosphates to a DNA chain (the primer strand). All DNA MgPolymerases2+ Require: DNAn + dNTP DNAn+1 + PPi This reaction constitutes a nucleophilic attack by the 3'-hydroxy group of the primer strand on the Templateα-phosphate strand of the incoming deoxynucleosidePrimer strand triphosphate. with a free 3’ OH This results2’-deoxynucleoside in the formation of a covalent 5’-triphosphates phosphodiesterMg2+ bond in a transesterification reaction. Note that polymerization proceeds in the 5"→3" direction. A driving force is the subsequent hydrolysis of PPi by inorganic pyrophosphatase. Requirements for DNA Replication by All DNA Polymerase Enzymes Template strand Primer strand with a free 3’ hydroxyl group 2’-deoxynucleoside 5’-triphosphates 2+ Mg 2 III. Prokaryotic DNA Polymerase Enzymes The first DNA polymerase to be isolated and characterized was DNA polymerase I from E. coli. (A. Kornberg; Nobel Prize, 1959) 2+ ProkaryoticPol I: 103 DNA kDa Polymerase polypeptide, Enzymes requires Mg , all four dNTP's, a DNA template strand and a primer strand with a 3’ hydroxyl group. The first DNA polymerase to be characterized was DNA polymerase I (Pol I) from E. coli. Pol I is a single 103 kDa polypeptide. Not essential for replication, but important in DNA repair The enzyme has three distinct catalytic activities: DNA Polymerase Activity 5' OH dNTP's OH + 5 PPi '3 5' Pol I is moderately processive; ~20 nucleotides are added per encounter with a DNA substrate at a rate of ~ 25 bp/sec The intrinsic error frequency of Pol I is less than 10-5 per base pair. Pol I also has two exonuclease activities: 3'->5' exonuclease => editing function (fidelity in DNA replication; 10-8 error rates) 5'->3' exonuclease => replication function and repair function Exonuclease Activity 1 '5 OH 3'->5' '5 OH + 5 dNMP '3 5' '3 5' Exonuclease Activity 2 '5 OH 5'->3' PO + 5 dNMP '3 5' '3 5' Pol I is NOT essential for cell replication. 3 III.III. ProkaryoticProkaryotic DNADNA PolymerasePolymerase Enzymes TheThe firstfirst DNADNA polymerasepolymerase to be isolated and characterizedcharacterized waswas DNADNA polymerasepolymerase II fromfrom E. coli. (A. Kornberg; NobelNobel Prize,Prize, 1959)1959) PolPol I:I: 103103 kDakDa polypeptide,polypeptide, requires Mg2+, allall fourfour dNTP's,dNTP's, aa DNADNA templatetemplate strandstrand andand aa primer strand with a 3’3’ hydroxylhydroxyl group.group. III. Prokaryotic DNA Polymerase Enzymes The first DNA polymerase to be isolated and characterized was DNA polymerase I from E. coli. (A. Kornberg; Nobel Prize, 1959) TheThe enzymeenzyme hashas threethree distinct catalytic activities: Pol I: 103 kDa polypeptide, requires Mg2+, all four dNTP's, a DNA template strand and a primer strand with a 3’ hydroxyl group. DDNNAA PPoollyymmeerraassee AAccttiivviitty 5' OH dNTP's OH 5' OH dNTP's OH ++ 5 5 P PPPi '3 5' i '3 5' Pol I is moderately processive; ~20 nucleotides are added per encounter with a DNA Pol I is moderately processive; ~20 nucleotides are added per encounter with a DNA substrate at a rate of ~ 25 bp/sec substrate at a rate of ~ 25 bp/sec Pol I has 3 distinct catalytic activities The enzyme has three distinct catalytic activities: The intrinsic error frequency of Pol I is less than 10-5 per base pair. The intrinsic error frequency of Pol I is less than 10-5 per base pair. DNA Polymerase Activity Pol I also has5' two exonucleaseOH activitiesdNT:P 's OH Pol I also has two exonuclease activities: + 5 PPi '3 5' 3'->5' exonuclease => editing function (fidelity in DNA replication; 10-8 error rates) 3'->5' exonucleaseReplication function: => editing function (fidelity in DNA replication; 10-8 error rates) Pol I is• moderatelyModerate processive processivity; ~20(adds nucleotides ~20 bp per are encounter added per with encounter DNA) with a DNA 5'->3'substrate exonuclease• Moderate at a rate of=>rate ~ 25replication (adds bp/sec ~25 bp function/sec) and repair function 5'->3' exonuclease• Moderate =>fidelity replication (~1 error in function 105 bp = ~10and-5 errors/repairbp function) -5 The intrinsicExonuclea serrore Acti vfrequencyity 1 of Pol I is less than 10 per base pair. Exonuclease Activity 1 '5 OH 3'->5' '5 OH Pol I also has two exonuclease activities: + 5 dNMP 3'->5' '5'3 O5'H ''35 OH 5' -8 + 5 dNMP 3''3-Editing>5' exonuclease function: => editing5' function (fidelity'3 in DNA replication; 10 error5' rates) • Boosts fidelity to ~10-8 errors/bp) 5'E-x>3'onu cexonucleaselease Activity 2 => replication function and repair function Exonuclease Activity 2 OH 5'->3' '5 PO Exonuclease Activity 1 + 5 dNMP '5'3 O5'H 5'->3' '3 5' PO '5 OH 3'->5' '5 OH + 5 dNMP '3 5' '3 + 5 dNMP 5' '3 5' '3 5' Repair function Pol I is NOT essential for cell replication. Pol I is NOT esseEntialxonucleas efor Activi tycell 2 replication. '5 OH 5'->3' PO + 5 dNMP '3 5' '3 5' Pol I is NOT essential for cell replication. 3 3 3 DNA Polymerase III is the enzyme responsible for genomic DNA (replication). Pol III is a multiproteinDNA Polymerase complex composed III of replicates 8 different subunits. DNA for cell replication A large, multiprotein complex composed of 8 different DNAsubunits replication by Pol III is extremely fast (1000 sec-1), highly processive (1000's bases added in a single encounter) and very accurate (<10-7 to 10-8 error rate).