Lagging Strand Synthesis

the ability to make corrections on the fly. Mismatches are sensed because they cause minor distortions in the shape of the double helix. The polymerase halts upon sensing a mismatch and removes the last nucleotide added. As this was added to the 3'-end of the growing DNA strand, the enzyme activity that removes the offending nucleotide is a 3'-exonuclease. In the case of DNA polymerase III, proofreading is due to a separate subunit, the DnaQ protein (e-subunit). (In some other DNA polymerases, proofreading ability resides on the same protein as polymerase activity.) In addition, immediately after replication, the new DNA is checked and if necessary repaired by the mismatch repair system (see Ch. 14).

One of the new strands of DNA, the "lagging strand", is made in short fragments that are joined up later.

The Complete Replication Fork Is Complex

The replication fork is defined as all the structural components in the region where the DNA molecule is being duplicated. It includes the zone where the DNA is being untwisted by gyrase and helicase, together with the stretches of single stranded DNA

3'-exonuclease An enzyme that degrades nucleic acids from the 3'-end mismatch Wrong pairing of two bases in a double helix of DNA

mismatch repair DNA repair system which recognizes and corrects wrongly paired bases

The Complete Replication Fork Is Complex 113

FIGURE 5.13 DNA Polymerase III— Proofreading

The portion of DNA polymerase responsible for checking the accuracy of new nucleotides is the DnaQ protein (e-subunits). A mismatch is detected by a bulge in the new chain. The incorrect nucleotide is discarded and the appropriate nucleotide added.

FIGURE 5.13 DNA Polymerase III— Proofreading

The portion of DNA polymerase responsible for checking the accuracy of new nucleotides is the DnaQ protein (e-subunits). A mismatch is detected by a bulge in the new chain. The incorrect nucleotide is discarded and the appropriate nucleotide added.

Molecular Biology Rarity

Both the tau (t) and gamma (g) subunits of DNA polymerase III are encoded by the same gene—dnaX. The tau subunit is a normal, full-length product of translation. However, the gamma subunit is made by frameshifting (see Ch. 13) followed by premature termination during translation of the same mRNA. Such differential synthesis of two proteins from a single gene is extremely rare in living cells, but this particular case appears to be widespread among bacteria, including E. coli. However, frameshifting to generate two alternative proteins is not so rare among RNA viruses with small genomes. For example, both retroviruses (e.g., HIV) and filoviruses (e.g., Ebolavirus) use this approach—see Ch. 17.

held apart by single strand binding protein (SSB). It also includes two molecules of DNA polymerase III, which are making two new strands of DNA (Fig. 5.14).Although the leading strand is made continuously, the lagging strand is made in short segments of 1,000 to 2,000 bases in length, known as Okazaki fragments after their discoverer.

Okazaki fragments The short pieces of DNA that make up the lagging strand

FIGURE 5.14 Components of the Replication Fork

The basic components of the replication fork are the DNA gyrase, DNA helicase, DNA polymerase III, and single strand binding proteins (SSB).

SSB pro Leios

DNA helicase

DNA gyrase

Parental DNA

As noted above, during DNA replication, the two new strands must be synthesized in opposite directions. A linear representation of this would imply that the two polymerase assemblies might move apart (Fig. 5.15). In fact, the two molecules of Pol III that are making these two strands are held together by the tau subunits. In order for them both to make new DNA simultaneously, the strands of DNA must be bent or looped, perhaps as shown in panel C of Figure 5.15.

Discontinuous Synthesis of DNA Requires a Primosome

Although the leading strand is synthesized continuously, the lagging strand is composed of multiple pieces, the Okazaki fragments. When synthesis of each new Okazaki fragment is begun, it needs a fresh RNA primer. For priming to occur, PriA protein displaces SSB proteins that are bound to the unpaired DNA from a short stretch of DNA and enables the primase (DnaG) to bind. The primase then makes a short RNA primer of 11 to 12 bases.This priming complex is sometimes known as the primosome (Fig. 5.16).

Each time a new Okazaki fragment is begun, the Pol III assembly that is making the lagging strand releases its grip on the DNA and relocates to start making a new strand of DNA starting from the 3' end of the RNA primer. This involves disassembly and relocation of the sliding clamp, which is performed by the clamp loading complex. Note that the replisome contains two Polymerase III assemblies, each with its own

PriA Protein of the primosome that helps primase bind primase Enzyme that starts a new strand of DNA by making an RNA primer primosome Cluster of proteins (including PriA and primase) that synthesizes a new RNA primer during DNA replication

Discontinuous Synthesis of DNA Requires a Primosome 115

A) TWO DNA Pol III SUBUNITS ACT TOGETHER

Direction of synthesis

Direction of synthesis

B) THE TWO SUBUNITS WOULD MOVE APART IF DNA WERE UNLOOPED

m jjj

C) LOOPING OF DNA ALLOWS SUBUNITS TO STAY TOGETHER

Pol III

Helicase

Pol III

Helicase

Imimy

Direction of synthesis

FIGURE 5.15 Relative Location of the Subunits of DNA Polymerase III at the Replication Fork

Imimy

Direction of synthesis

FIGURE 5.15 Relative Location of the Subunits of DNA Polymerase III at the Replication Fork

Antiparallel synthesis dictates that the DNA polymerase III assemblies should change their relative positions as DNA synthesis occurs (compare A with B). Since the two assemblies are held by tau subunits, they cannot in fact move apart. Panel C suggests that the DNA is looped around to allow the DNA polymerase components to remain in contact.

FIGURE 5.16 Three Steps in Starting a Primer for a New Okazaki Fragment

Prior to primer formation, the bases of the parental DNA strand are covered with SSB proteins. A) First, the PriA protein displaces the SSB proteins. B) Second, a primase associates with the PriA protein. C) Lastly, the primase makes the short RNA primer needed to initiate the Okazaki fragment.

Parental DNA

Parental DNA

A) PriA DISPLACES SSB PROTEIN PriA

S1 51

B) PRIMASE BINDS

B) PRIMASE BINDS

C) PRIMASE MAKES SHORT RNA PRIMER

Primosome

Primosome

FIGURE 5.16 Three Steps in Starting a Primer for a New Okazaki Fragment

Prior to primer formation, the bases of the parental DNA strand are covered with SSB proteins. A) First, the PriA protein displaces the SSB proteins. B) Second, a primase associates with the PriA protein. C) Lastly, the primase makes the short RNA primer needed to initiate the Okazaki fragment.

The fragments of the discontinuous lagging strand must be linked by removing the RNA primers, filling the gaps with DNA and, finally, joining the ends.

sliding clamp, but only a single clamp loader. This is because only the lagging strand needs constant clamp removal and reloading. It is worth repeating that in E coli, all of this happens about 1,000 times per second.

Completing the Lagging Strand

After the replication fork has passed by, the lagging strand is left as a series of Okazaki fragments with gaps (that is, spaces from which one or more nucleotides are missing)

gap A break in a strand of DNA or RNA where bases are missing

Completing the Lagging Strand 117

Okazaki RNA primer Okazaki

Okazaki RNA primer Okazaki

nucleotides

FIGURE 5.17 Three Steps in Joining the Okazaki Fragments

When first made, the lagging strand is composed of alternating Okazaki fragments and RNA primers. The first step in replacing the RNA primer with DNA is the binding of DNA polymerase I to the primer region. As Pol I moves forward it degrades the RNA and replaces it with DNA. Lastly, DNA ligase seals the nick that remains.

Parental DNA

DNA ligase

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Nick

Nick sealed

Parental DNA

between them. The gaps are filled with the RNA primer. Joining the Okazaki fragments to give a complete strand of DNA is accomplished by two—or perhaps three— enzymes working in succession: Ribonuclease H (RNase H), DNA polymerase I (Pol I), and DNA ligase. Only the last two enzymes are involved in the classical model. DNA polymerase I both degrades the RNA primers and fills the gaps left by the degraded RNA. Finally, DNA ligase joins the ends (Fig. 5.17). It has been suggested that in fact RNase H, which degrades the RNA strand of DNA:RNA double helixes, removes most of each RNA primer and that DNA polymerase I only removes the last few bases of the RNA primers.

Despite being a single polypeptide chain, DNA polymerase I possesses kinetic proofreading ability like Pol III. Pol I also has the unique ability to start replication at a nick in the DNA. The term "nick" refers to a break in the nucleic acid backbone with no missing nucleotides. When Pol I finds a nick, it cuts out a small stretch of DNA—or RNA—approximately 10 bases long. It then fills in the gap with new DNA. Pol I is important in completing the lagging strand as well as in DNA repair (see Ch.

DNA ligase Enzyme that joins up DNA fragments end to end

DNA polymerase I (Pol I) Bacterial enzyme that makes small stretches of DNA to fill in gaps between Okazaki fragments or during repair of damaged DNA

nick A break in the backbone of a DNA or RNA molecule (but where no bases are missing)

ribonuclease H (RNase H) Enzyme that degrades the RNA strand of DNA:RNA hybrid double helixes. In bacteria it removes the major portion of RNA primers used to initiate DNA synthesis.

FIGURE 5.18 Sequences at the Origin of DNA Replication

Sequence repeats at the origin of replication are of two varieties, both being AT-rich.

13 bp repeats GATCTNTTNTTTT

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