Replication of DNA Lecture 7 W3310/4310 Spring 2013 It’s all about Initiation

• Problems faced by DNA replication machinery • must replicate their genomes to make new progeny

2 Virus Genomes Require Special Copying Mechanisms

Parvovirus Herpesvirus

Adenovirus Polyomavirus

3 DNA Replication

• Replication requires expression of at least one virus protein, sometimes many • DNA is always synthesized 5’ - 3’ via semiconservative replication • Replication initiates at a defined origin using a primer • The host provides other proteins

4 What’s the Host for? Viruses Can’t do it Themselves

• Viruses are parasites • Enzymes and scaffolds • Simple viruses conserve genetic information - always hijack more host proteins • Complex viruses encode many, but not all proteins required for replication

5 DNA Replication

• Genomes come in a wide assortment of shapes and sizes • Replication yields progeny - a switch for regulation - an important regulatory event • Always delayed after infection

6 Outcomes of DNA Replication

• Lytic infection - new progeny - high copy number • Latent infection - stable assimilation in host at low copy number - virus genomes may be episomal or integrated

7 Requirements for DNA Replication

• Ori recognition for initiation - binding to an AT-rich DNA segment • Priming of DNA synthesis - RNA - Okazaki fragments - DNA - hairpin structures - protein - covalently attached to 5’ end • Elongation • Termination

8 Requirements for DNA Replication

• Viruses don’t replicate well in quiescent cells • Induction of host replication enzymes and cycle regulators • Virus encoded immediate early and early gene products

9 Where Does the Polymerase Come From?

• Small DNA viruses do not encode an entire replication system -encode proteins that orchestrate the host -, , • Large DNA viruses encode most of their own replication systems -, ,

10 Virus Encoded Proteins

• Origin Binding Protein, Helicases and Primase • DNA polymerase and accessory proteins • Exonucleases • Thymidine kinase, RR, dUTPase

11 Replication Occurs at Replication Centers! • DNA templates and rep proteins • Form at discrete sites ND10’s (PML bodies) • Polymerases, ligases, helicases, topoisomerases

Pre

Post

12 Getting Started at Viral Origins

• AT-rich DNA segments recognized by viral origin recognition proteins - seed assembly of multiprotein complexes • Some viruses have one ori others up to three - used for different purposes • Often associated with transcriptional control regions

13 Virus Genomes

TR TR p5 p19 p40

Ori

14 How to supercoil DNA

15 What Do Oris Look Like?

16 Origin Recognition Proteins

• Polyoma Tag binds specifically to DNA • Papilloma E1 binds to ori in presence of E2 • AAV Rep68/78 binds at ends and unwinds DNA, also involved in terminal resolution • Adenovirus pTP binds at terminus and recruits DNA polymerase • Herpesvirus UL9 protein recruits viral proteins to AT-rich ori’s and then unwinds DNA

17 dsDNA Virus Genomes

18 ssDNA Genomes

Parvoviridae

ITR ITR 19 Hepadnavirus -Cycle

What to do with a molecule that looks this way

20 Two Basic Modes of Replication

Leading Replication Fork Strand displacement (primer) 5’

5’ 3’ Primer 3’ 3’ 5’ Papillomaviruses Polyomaviruses Adenoviruses (protein) Herpesviruses Parvoviruses (DNA hairpin) Retroviral Poxviruses (hairpin Proviriuses Lagging

21 Polyomavirus Replication Forks

• Initiation from a single ori, requires expression of Tag • Replicate as covalently closed circles • Leading strand replication occurs via extension from an RNA primer • Lagging strand replication is delayed until the replication fork has moved - also uses RNA primers - creates discontinuities • How to fill in the gaps?

22 Properties of T • T is a species-specific DBP/OBP -preinitiation complexes do not form in the wrong species -failure to interact with DNA polα-primase • Binds and sequesters cell cycle regulators -causes cells to enter S phase - WHY? • T synthesis is autoregulated -protein is heavily modified -controls DNA binding -promotes cooperativity -affects unwinding of DNA 23 T antigen a multifunctional virus-specified

24 What’s the Ori Core Sequence?

Late promoter

Between pE and pL LT binding sites, SP1 sites AT rich Nucleosome free - Why? 25 Polyomaviruses • Covalently closed circular, double stranded DNA

Bidirectional replication

Leading Lagging

Lagging 26 Leading Leading vs. Lagging

• Leading strand DNA synthesis is continuous • Lagging strand DNA synthesis is discontinuous • Direction of synthesis off of either template strand is the same

27 Initiation of DNA Synthesis LT Binding A/T site II EP

1 ATP + LT

Two LT hexamers bind

28 Initiation of DNA Synthesis

Conformational change 2 of EP sequence

Binding distorts early palindrome unwinding origin

29 Initiation of DNA Synthesis

ATP 3 RPA ADP + Pi

Binding of Rpa occurs

30 Initiation of DNA Synthesis

Two LT hexamers bind

Binding distorts early palindrome unwinding origin

Binding of Rpa occurs

31 DNA Synthesis Initiates at a Unique Origin Ori RE Site Ori RE Site

RE Site

How do you know that replication is bidirectional?

32 The Problem How to connect the Okazaki fragments

33 The Leading Strand Is Easy

• Presynthesis complex α, T and Rp-A • Rf-C binds 3’OH along with PCNA and pol δ -Rf-C a clamp loading protein -Allows entry of PCNA on DNA -Causes release of pol α • Form sliding clamps along DNA • Continuous copying of parental strand

34 The Lagging Strand - Not So Easy • 1st primer and Okazaki fragment made by pol α-primase complex • DNA is copied from the replication fork toward the origin • Multiple initiations are required to replicate the template strand • Both leading and lagging strands move in the same direction! • Which moves, the DNA or the complex? -Template has to move, otherwise...... ?

35 Unwinding at the Ori

SSBP

36 Cellular Proteins Required for Polyomavirus DNA Replication

37 DNA Synthesis by Polyomaviridae is Bidirectional

Leading strand presynthesis complex

38 DNA Synthesis by Polyomaviridae is Bidirectional

Rf-C binds 3’ R-D Lagging strand pcna is next Polα/primase

PCNA RFC

39 DNA Synthesis by Polyomaviridae is Bidirectional

40 DNA Synthesis by Polyomaviridae is Bidirectional

Remove RNA, fill gaps, seal

41 Polyomaviridae DNA Synthesis

Leading strand presynthesis complex

Rf-C binds 3’ R-D Lagging strand pcna is next

Remove RNA, fill gaps, seal 42 The DNA Replication Machine

Lagging strand

Leading strand

43 The Replication Machine

http://www.hhmi.org/biointeractive/media/ DNAi_replication_vo1-lg.wmv

44 Problems in Replication

Catenated molecules

45 Termination - the End

• Separate daughter molecules from replication complex • Topos relax and unwind supercoils - relieve torsional stress caused by unwinding - unwinding leads to overwinding throughout the rest of the molecule • Topo II decatenates - separating daughter molecules by cleaving and resealing the replicated molecules

46 DNA Priming • Priming via a specialized structure • Parvoviruses self prime, form a template primer • Their genomic are ss of both + and - polarity and they contain Inverted Terminal Repeats

start here - but how to get to the end?

47 AAV Replication is Continuous ITR

• A dependovirus! • No pol α, uses Inverted Terminal Repeat to self-prime • Requires pol δ, Rf-C and PCNA • Rep78/68 proteins are required for initiation and resolution - endonuclease, helicase, binds 5’ terminus • No replication fork!

48 Replication of Adenovirus

• Strand displacement synthesis • Utilizes a protein primer • Origins at both ends • Assembly of pTP into a preinitiation complex activates covalent linkage of dCMP to a S residue in pTP by viral DNA pol • Semiconservative DNA replication from different replication forks

49 Protein Priming

• Adenoviridae - -Precursor to terminal protein (pTP) -Ad DNA pol links α-phosphoryl of dCMP to OH of a S residue in pTP -Added only when protein primer is assembled with DNA pol into preinitiation complex at ori -3’OH primes synthesis of daughter strand

50 Protein Priming

Displaced ss Template DNA

ITRs Single-stranded DNA Template 51 Virus

• HSV 2 oriS and a unique oriL sequence • Four equimolar isomers of virus genome • DNA enters as linear molecule converts to circle • Virus dissociates host ND10s • Replicates as a rolling circle

52 Initiation of Herpesvirus DNA Replication

UL US

Circularization DNA ligase IV/ XRCC4

Host proteins are responsible for circularization

53 HSV Gene Products Required for Replication • UL5, 8 and 52 - form primase • UL42 - a processivity protein • UL9 - Origin Binding Protein • UL29 - SS DNA Binding Protein • UL30 - DNA polymerase • Necessary but not sufficient!

54 Herpesvirus DNA Replication

OBP ss DBP ss DBP Helicase-Primase

HSV Polymerase Processivity Protein

55 Rolling Circle Replication

Cleavage Packaging Sites

56