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.

Requirements for DNA Replication by All DNA Polymerase Enzymes

Template strand Primer strand with a free 3’ hydroxyl group 2’-deoxynucleoside 5’-triphosphates Mg2+

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 Mg2+

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 Mg2+

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) The enzyme has three distinct catalytic activities: The enzyme has three distinct catalytic 2+activities: Pol I: 103 kDa polypeptide, requires Mg , 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) • Moderate processivity (adds ~20 bp per encounter with DNA) Pol I is moderately processive; ~20 nucleotides are added per encounter 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 -5 5'->3' exonuclease• Moderate =>ﬁdelity replication (~1 error in function 10 bp = ~10and 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 '5'3 O5'H 3'->5' ''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). • Very fast: ~1000 bp/sec The enzymes possess 3'->5' exonuclease activity, but not 5’->3’ exonuclease activity. • Very processive: 1000 bp/encounter • Possesses 3’ 5’ exonuclease activity, but not 5’ 3’

IV. •DNA Polymerase Replication in ﬁdelity: E. coli. Complex~10-5 errors/ biologicalbp processes, boosted typically to 10 ha-7ve-10 discrete-8 initiation,by elongation exonuclease and termination proofreading steps.

Initiation Phase. Replication of chromosomal DNA begins at a discrete site known as the origin of replication (in E. coli, this is known as oriC).

Ori C Binding of dnaA protein to oriC results in dnaA Protein unwinding of the DNA duplex. ATP

The duplex is further unwound with the binding of dnaB and dnaC proteins. dnaB & dnaC Proteins ATP The unwound DNA is stabilized and protected with the binding of single-stranded binding protein (SSB). SSB ATP A bubble forms at oriC with the unwinding of DNA occurring in both directions.

4 DNA Polymerase III is the enzyme responsible for genomic DNA (replication). Pol III is a multiprotein complex composed of 8 different subunits.

DNA 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).

The enzymes possess 3'->5' exonuclease activity, but not 5’->3’ exonuclease activity.

IV. DNA Replication in E. coli. Complex biological processes typically have discrete initiation, elongation and termination steps.

The DNA Replication Process in E. coli Initiation Phase. Replication of chromosomal DNA begins at a discrete site known as Initiationthe origin Phaseof replication. Replication (in E. coli, ofthis chromosomal is known as oriC DNA). begins at a discrete site known as the origin of replication (in E. coli, this is known as oriC). Ori C Binding of dnaA protein to oriC results in dnaA Protein unwinding of the DNA duplex. ATP

4 The DNA Replication Process in E. coli

Primase is an RNA polymerase that binds to single-stranded DNA in a multiprotein Primase complex known as a primosome (seven proteins).

Primase synthesizes short (~ 5 – 10 base) rNTP's RNA primers.

PPi Primase dissociates and Pol III utilizes these 5' (RNA)

RNA primers for the synthesis of DNA. 5' (RNA)

5' Elongation Phase. Assembly of pol III 5' complex onto duplex DNA results in the Primase formation of a "replication fork". HO 5'

-5 5' OH Polymerase error rate ~ 10 DNA Pol III

3’->5’ exonuclease corrects > 99% HO 5' -7 -8 => 10 to 10 overall error rate 5' OH

dNTP's

PPi DNA Synthesis

DNA Gyrase Remember “Leading Strand” vs. “Lagging Strand” synthesis and Okazai Fragments …

Positive supercoiling induced by unwinding of duplex DNA is prevented by DNA gyrase; this enzyme introduces negative supercoils as it hydrolyzes ATP.

5 Primase is an RNA polymerase that binds to single-stranded DNA in a multiprotein Primase complex known as a primosome (seven proteins).

Primase synthesizes short (~ 5 – 10 base) rNTP's RNA primers.

PPi ThePrimase DNA dissociates Replication and Pol III utilizes theseProcess in E. coli 5' (RNA)

RNA primers for the synthesis of DNA. 5' (RNA)

5' Elongation Phase. Assembly of pol III 5' complex onto duplex DNA results in the Primase formation of a "replication fork". HO 5'

-5 5' OH Polymerase error rate ~ 10 DNA Pol III

3’->5’ exonuclease corrects > 99% HO 5' -7 -8 => 10 to 10 overall error rate 5' OH

dNTP's

PPi DNA Synthesis

DNA Gyrase Remember “Leading Strand” vs. “Lagging Strand” synthesis and Okazai Fragments …

Positive supercoiling induced by unwinding of duplex DNA is prevented by DNA gyrase; this enzyme introduces negative supercoils as it hydrolyzes ATP.

5 Primase is an RNA polymerase that binds to single-stranded DNA in a multiprotein Primase is an RNA polymerase that binds to Primase complex known as a primosome (seven Primasesingle is-stranded an RNA DNA polymerase in a multiprotein that binds to proteins). Primase singlecomplex-stranded known DNA as a in primosome a multiprotein (seven Primase complexproteins) known. as a primosome (seven Primaseproteins) synthesizes. short (~ 5 – 10 base) rNTP's RNA primersPrimase synthesizes. short (~ 5 – 10 base) rNTP's Primase synthesizes short (~ 5 – 10 base) PPi RNA primers. rNTP's RNA primers. Primase dissociates and Pol III utilizes these PPi 5' (RNA) PPi RNA primersPrimase dissociatesfor the synthesis and Pol III of util DNA.izes these 5' (RNA) 5' Primase dissociates and Pol III utilizes these (RNA) 5('RNA) RNA primers for the synthesis of DNA. (RNA) 5' (RNA) RNA primers for the synthesis of DNA. 5'

Elongation Phase. Assembly of pol III 5' 5' Elongation Phase. Assembly of pol III 5' Elongation Phase. Assembly of pol III 5' complex onto duplex DNA results in the 5' complex onto duplex DNA results in the Primase complex onto duplex DNA results in the Primase formation of a "replication fork". Primase formation of a "replication fork". HO 5' formation of a "replication fork". HO 5' HO 5' -5 5' OH 5' OH Polymerase error rate ~ 10 -5- 5 5' OH DNA Pol III Polymerase error rate ~ 10 DNA Pol III Polymerase error rate ~ 10 DNA Pol III

3’->5’ exonuclease corrects > 99% HO 5' HO 3’->5’3’-->5’7 exonuclease exonuclease-8 corrects corrects > > 99% 99% HO 5' 5' -7 -7 -8 -8 5' OH => 10 to 10 overall error rate 5' => 10 to 10 overall error rate 5' OH OH The => 10 DNA to 10 overallReplication error rate Process in E. coli dNTP's dNTP'sdNTP's

PP PP PPi i i DNADNA SynthesisDNA Synthesis Synthesis

DNA Gyrase DNA Gyrase DNA Gyrase RememberRemember “Leading “Leading Strand” Strand” vs. vs. “Lagging “Lagging RememberStrand” “Leading synthesis Strand”and Okazai vs. Fragments “Lagging … Strand” synthesisStrand” synthesis and Okazai and Okazai Fragments Fragments …

Okazaki fragments

Positive supercoiling induced by unwinding of Positiveduplex DNAsupercoiling is prevented induced by by DNA unwinding gyrase of; this duplexenzymePositive DNA introduces supercoilingis prevented negative by inducedDNA supercoils gyrase by; thisunwindingas it of enzymehydrolyzesduplex introduces DNA ATP. is negative prevented supercoils by DNA as it gyrase; this hydrolyzesenzyme ATP. introduces negative supercoils as it hydrolyzes ATP.

5 5 5 The DNA Replication Process in E. coli

Termination of DNA Replication

Replication of the E. coli genome ends when the replication forks meet at the termination region (ter).

The “concatanes” are resolved by topoisomerase IV.

Fluoroquinolone antibiotics inhibit both DNA gyrase (gram negative) and topo IV (gram positive) enzymes.

DNA Mismatch Repair.

DNA polymerases Ciproﬂoxacindo not make mistakes, Levoﬂoxacin but when they do, 99% of them will be fixed by the DNA Mismatch Repair system; this system reduces the overall replication error rate to 10-9 to 10-10.

E. coli genome ~ 5 x 106 bp; Human genome ~ 3 x 109

6 Termination of DNA Replication

Replication of the E. coli genome ends when the replication forks meet at the termination region (ter).

The “concatanes” are resolved by topoisomerase IV.

Fluoroquinolone antibiotics inhibit both DNA gyrase (gram negative) and topo IV (gram positive) enzymes.

DNA Mismatch Repair.

DNA polymerases do not make mistakes, but when they do, 99% of them will be fixed by the DNA Mismatch Repair system;DNA this system Mismatch reduces the Repair overall replication error rate to 10-9 to 10-10.

This system ﬁxes >99% of the rare errors made by DNA polymerases, boosting the overall ﬁdelity to 10-9 to 10-10 errors/bp

(E. coli genome: ~5x106 bp Human genome: ~3x109 bp)

E. coli genome ~ 5 x 106 bp; Human genome ~ 3 x 109

6 DNA Damage Repair

UV light, X-rays, reactive oxygen species (ROS), plant toxins, chemical carcinogens,DNA Damage and Repai therapeuticr. UV light, X- rays,alkylating reactive oxygen agents species can (ROS), damage plant toxins, DNA. chemical carcinogens, and therapeutic alkylating agents can damage DNA.

Base Excision Repair Nucleotide Excision Repair

Plasmids are circular extra-chromosomal duplex DNA molecules that replicate autonomously; the number of plasmid molecules per cell is determined by the “strength” of the replication origin.

Plasmids may be transferred between bacteria and often carry genes responsible for antibiotic resistance and toxins.

7 DNA Damage Repair. UV light, X-rays, reactive oxygen species (ROS), plant toxins, chemical carcinogens, and therapeutic alkylating agents can damage DNA.

Base Excision Repair Nucleotide Excision Repair

Bacterial Plasmids

Circular, extra-chromosomal, double-stranded DNA molecules that replicate autonomously PlasmidsThe number are circular of plasmidextra-chromosomal molecules duplex per DNA cell molecu is lesdetermined that replicate by the autonomously; the number of plasmid molecules per cell is determined by the “strength”“strength” of the of replication the replication origin. origin Plasmids may be transferred between bacteria and often Plasmids may be transferred between bacteria and often carry genes responsible for carryantibiotic genes resistance responsible and toxins. for antibiotic resistance and toxins

7 DNA Replication in Eukaryotes

The process is similar, but more complex; there are at least 13 different eukaryotic DNA polymerases (named with Greek letters).

DNA polymerase α:primase complex synthesizes an RNA primer and then adds ~ 15 bases of DNA. This primer is removed by FEN-1 nuclease.

DNA pols δ and ε are the main polymerases involved in chromosomal replication.

Many of the other polymerases appear to function primarily in DNA repair pathways (e.g., pol β), plus those found in mitochondria (pol γ) and chloroplasts. V. Eukaryotic DNA Replication

The process is essentially identical, but more complex; there are at least 13 different eukaryotic DNA polymerases (named with Greek letters).

DNA polymerase-primase α synthesizes an RNA primer and then adds ~ 15 bases of DNA. This primer is removed by FEN-1 nuclease.

DNA polymerases δ and ε are the main polymerases involved in chromosomal replication.

DNA Replication in Eukaryotes

Camptothecin and Etoposide are topoisomerase poisons (Topo I and Topo II, respectively). They trap the topo:DNA complexes and ultimately cause single and double-strand DNA breaks in the duplex. They are used in cancer chemotherapy.

Many of the other polymerases appear to function primarily in DNA repair pathways (e.g., DNA polymerase β), plus those found in mitochondria (DNA polymerase γ) and chloroplasts.

Episomes are the eukaryotic equivalent of bacterial plasmids (herpesviruses …)

Camptothecin and Etoposide are topoisomerase poisons (Topo I and Topo II, respectively). They trap the topo•DNA complexes and ultimately cause single and double-strand DNA breaks in the duplex. They are used in cancer chemotherapy.

8 Bottom Line

Know the requirements for DNA synthesis and how nucleotides are added to the primer strand (i.e.- what is the chemical reaction that takes place) and what makes the reaction “irreversible”. Know the multiple catalytic activities of pols I and III and their function in DNA replication and repair. Understand the meaning of processivity and ﬁdelity in DNA synthesis. Know and be prepared to discuss the ﬁdelity and processivity of pol I vs. pol III and how these features are well suited to their biological function. Be prepared to describe the general features of initiation, replication and termination of genomic replication in E. coli. What roles do gyrase and topoisomerase IV play? What is the arrangement of DNA in a replication fork? Be prepared to describe the essential features of DNA mismatch repair and DNA damage repair. You should know what a plasmid is and why they are important to bacteria. Know the common features between prokaryotic and eukaryotic DNA replication. Know the correct spelling for any and all drugs and diseases discussed during the semester and the biochemistry behind them