The Replication of DNA II

DNA polymerases are usually poor at separating the two base paired strands of dublex dNA. So, at the ______ ______, a third class of enzyme called ____ ______, catalyze the separation of the two strands of duplex DNA.

DNA helicases ____ to and ____ directionally along ssDNA using the energy of ______ _______ (usually ______) binding and ______ to displace any DNA strand that is annealed to the bound ssDNA

Typically, DNA helicases that act at replication forks are ______ proteins that assume the shape of a _____. These ____ shaped protein complexes encircle one of the two _____ _____ at the replication fork adjacent to the single stranded:double stranded junction.

Like DNA polymerases, DNA helicases act _________, each time thy associate with substrate, they ______ multiple base pairs of DNA.

Why do DNA helicases have high processivity? What happens to the helicase when it reaches the end of the DNA strand that it has encircled

The arrangement of DNA helicase and DNA poses problems for the binding of the DNA helicase to the DNA substrate. Where is this most obvious and why?

Due to the reality that DNA helicases are almost always loaded on the DNA at ______ sites, of _____ chromosomes, the same problem exists during the ______ of these DNAs. Thus there are specialized ______ that open the DNA helicase ring andplace it around the DNA before ______ the ring.

This topological linkage between proteins involved in DNA replication and their DNA substrates is a common mechanism to increase ________.

Each DNA helicase moves along ______ in a defined direction. This property is referred to as the _____ of the DNA helicase.

DNA helicases can have a polarity of either ___ to ___ or ___ to ___. This direction is always defined according to the strand of DNA bound as opposed to the strand that is ______.

In the case of DNA helicase that functions on the ______ strand template of the rep. for, the polarity is ___ to ___ to allow the DNA helicase to proceed toward the duplex region of the rep. fork.

As is true for all enzymes that move along DNA in a directional manner, movement of the ______ along ssDNA requires the input of ______ energy. For helicases, this energy is provided by ____ _______

DNA helicase pulls single stranded DNA through a _____ ______ ______. Single stranded DNA binding proteins _______ ssDNA before replication

After the DNA helicase has passed, the newly generated ssDNA must remain free of _____ ____ until it can be used as a template for DNA synthesis. To stabilize the separated strands, _____ ______ _______ rapidly bind to the separated strands.

Binding of one SSB promotes the binding of ______ ____ to the immediately adjacent ______. This is called _______ ______ and occurs because SSB molecules bound to immediately adjacent regions also bind to _____ ____.

This SSB-SSB interaction strongly ______ SSB binding to ssDNA and makes sites already occupied by one or more SSB molecules preferred ____ binding sites

Cooperative binding ensures that ssDNA is rapidly coated by _____ as it emerges from the DNA helicase. Once coated, ssDNA is held in an ______ state that facilitates its use as a template for DNA or RNA _____ ______

SSBs interact with ssDNA in a ______ ______ manner. SSBs primarily contact ssDNA through ________ interactions with the _______ backbone and _______ interactions with the DNA bases. In contrast to sequence specific DNA binding proeins, SSBs make few if any ______ ______ to the ssDNA bases

__________ remove supercoils produced by DNA unwinding at the rep. fork

As the strands of DNA are separated at the replication fork, the dsDNA in front of the for becomes increasingly ________ supercoiled. What causes this?

If the DNA strands remain unbroken, ther can be no reduction in ______ ______ (define) to accommodate this unwinding of the DNA duplex. Thus, as the DNA helicase proceeds, the DNA must accommodate the same ______ ______ within a ______ number of base pairs.

Indeed, for the DNA in front of the rep. fork to remain relaxed, one DNA link must be ________for every ~10bp of DNA _______.

If there were no mechanism to relive the accumulation of these supercoils, the replication machinery would grind to a ______ in the face of mounting ______ placed on the DNA in front of the rep fork

This problem is most clear for the ________ chromosomes of _______, but it also applies to euk. chromosomes. Because euk. chromosomes are not closed circles, they could in principle _______ along their length to dissipate the introduced supercoils. In reality, it is simply impossible to _______ a DNA molecule (millions of base pairs) each time a turn of the helix is _______

The supercoils introduced by the action of the DNA helicase are removed by _________ that act on the ________ dsDNA in front of the rep. fork. These enzymes do this by breaking either _____ or ______ strands of DNA without letting go of the DNA and passing the same number of DNA strands through the ______.

The action of topoisomerases relieves the accumulation of ________

On its own, DNA polymerase can only efficiently extend ______ primers  annealed to ssDNA templates. The addition of ______, ___ _______ and ___________ dramatically extends the possible substrates for DNA polymerase.

Primase provides the ability to initiate new DNA strands on any piece of ssDNA. Of course, the use of primase also imposes a requirement for the removal of the _____ _____ to complete replication.

Strand separation by DNA helicase and dissipation of ______ ______ by topoisomerase allow DNA polymerase to replicate dsDNA. This process is seen across a vast array of organisms to accomplish DNA rep., name 3

DNA helicase and topoisoemerse perform their functions without permanently altering the ______ structure of DNA or synthesizing any _____ molecules. DNA helicase breaks only the _______ bonds that hold the two strands of DNA together without breaking any _______ bonds

Although topoisomerases break one or two of the targeted DNA's _______ bonds, each bond broken is precisely ______ before the topoisomerase releases the DNA. Instead of altering the _______ structure of DNA, the actions of these enzymes results in a DNA molecule with an _______ conformation. Importantly, these conformational alterations are _______ for the duplication of the large dsDNA molecules that are the foundation of _______ and ______ chromosomes

The proteins that act at the rep fork interact tightly but in a sequence _________ manner with DNA. These interactions exploit of DNA that are the same regardless of the particular base pair. Name those features

The central role of DNA polymerases in the efficient and accurate replication of the genome has resulted in the evolution specialized _____ ________. For example, E. coli has at least _____ that are distinguished by their _______ properties, ______ composition, and _______.

____ ________ ___ is the primary enzyme involved in the replication of the chromosome in E. coli

Because the entire 4.6Mb E. coli genome is replicated by ____ replication forks, DNA pol III must be highly ________. Consistent with these requirements, DNA pol III is generally found to be part ofa large complex that confers very high ________. The complex is called _____ _____ _____ ________.

In contrast to DNA pol III holoenzyme, DNA polymerase I (DNA pol I) is specialized for the removal of the ____ ______ that are used to initiate DNA synthesis. For this reason, DNA polymerase has a ___ ________ that allows DNA pol I to remove RNA or DNA immediately upstream of the site of DNA synthesis.

Unlike DNA pol III holoenzyme, DNA pol I is not highly _______, adding only 20 - 100 nucleotides per binding event. These properties are ideal for _____ _______ removal and DNA synthesis across the resulting ssDNA ____.

The ___ _______ of DNA pol I can remove the RNA-DNA linkage that is resistant to ______ __. The low ________ of DNA pol I readily synthesizes across the short region previously occupied by an ____ _________ but is released before _______ and resynthesizing large amounts of DNA that was primed by the RNA.

When DNA pol i completes its function, only a _____ is present in the DNA. Because DNA pol I and DNA pol III are involved in DNA replication, both of these enzymes must be highly ________. Thus both proteins include an associated ________ ________. The remaining three DNA polymerases in E. coli are specialized for DNA ______ and ____ ________ activities

Eukk cells also have multiple DNA polymerases, with a typical cell having more than _____. Of these, ______ are essential to duplicate the genome, name them

Each of the essential DNA polymerases in Euk. cells is composed of multiple _________. DNA pol α/primase is specifically involved in initiating new _____ _____. This _____ subunit protein complex consists of a _____ subunit DNA pol α and a _____ subunit DNA pol primase.

After the primase synthesizes an _____ ______, the resulting RNA primer:template junction is imemediately handed off to the associated ____ _____ ____to initiate DNA synthesis

Because of its relatively _____ processivity, DNA pol α/primase is rapidly replaced by the ______ processive  DNA pol δ, and DNA pol ε.

The process of replacing DNA pol α/primase with DNA pol δ, and DNA pol ε is called _______ _______ and results in _____ different DNA polymerases functioning at the euk. ______ ______.

DNA pol δ, and DNA pol ε are specialized to synthesize different _____ at the rep fork, while DNA pol ε synthesizes the _____ _____ and DNA pol δ synthesizes the ______ ______. As in bacterial cells, the majority of the remaining euk. DNA polymerases are involved in _____ ______

High processivity at the rep fork ensure rapid duplication, but in the absence of other ______, DNA polymerases that act at the rep for are only able to synthesize 20 - 100 bp before releasing from the ______.

One key to the processivity of the DNA polymerases that act at rep forks is their association with proteins called ______ _____ _______. These proteins are composed of multiple _____ subunits that assemble in the shape of a ______.

The hole in the center of the clamp is large enough to _____ the DNA double helix and leave room for a layer of one or two _____ molecules between the DNA and the protein. These properties allow the clamp proteins to slide along the DNA without ________ from it

Sliding DNA clamps also bind tightly to ____ ______ bound to primer:template junctions. The resulting complex moves _______ along the DNA template during DNA synthesis.

In the absence of a sliding clamp,a DNA polymerase _______ and _______ away from the template DNA, on average, once every 20-100 bp synthesized. In the presence of the sliding clamp, the DNA polymerases still _________its active site from the _______ end of the DNA frequently, but the association with the sliding clamp prevents the polymerase from ______ away from the DNA

By keeping the DNA polymerase in _____ proximity to the DNA, the sliding clamp ensures that the DNA polymerase ______ rebinds to the _____ primer:template junction, vastly ________ the processivity of the DNA polymerase

Once an ssDNA template has directed synthesis of its complementary stand, the DNA polymerase must ______ from the completed dsDNA and the sliding clamp to act at a new _____ _____ _______. This release is accomplished by a change in the _______ between the DNA polymerase and the sliding clamp that depends on the bound DNA

DNA polymerase bound to a primer:template junction has a ______ affinity for the clamp. In contrast, when a DNA polymerase reaches the end of an ss DNA template (e.g. at end of an _______ _______), the presence of dsDNA in its active site resultsin a change in ______ that ______ the polymerase's affinity for the sliding clamp and the DNA.

When a polymerase completes the replication of a stretch of DNA, it is ______ from the sliding clamp so that it can act on a new _____ ______ _______.

Once released from a DNA polymerase, sliding clamps are not immediately ______ from the replicated DNA. Instead, other _______ that function at the site of rcent DNA synthesis interact with the ______ ______.

Enzymes that assmble chromatin in euk cells are recruited to the sites of DNA rep. by an interaction with the euk _______ ____ _____ (called PCNA). Similarly, euk proteins invloved in ______ ______ repair also interact with _____ _____ proteins. In each case, by interacting with the ____ _____, these proteins accumulate at sites of new DNA synthesis where they are ______ most

Sliding clamp proteins are a conserved part of the DNA rep. apparatus derived from organisms as diverse as ______, _______, _______ and ______. In each case, the clamp has a ______ symmetry and the same _______. The main difference is the number of ______ that come together to form different clamps.

The sliding clamp is a ______ ring in solution but must open to encircle the DNA double helix.  A special class of protein complexes called ______ _____ ______, catalyze the opening and placement of sliding clamps on the DNA.

Sliding clamp loaders couple ____ ______ and _________ to the placement of the slidng clamp around primer:template junctions on the DNA. The clamp loader also removes _____ ______ from the DNA when they no longer in use. **This does not require ____ ______

Like DNA helicases and topoisomerases, sliding clamp loaders alter the ______ of their target but not its _______ composition.

_______ of a sliding  clamp occurs anytime a primer:template junction is present in the cell. These DNA structures are formed not only during DNA replication, but also during several ____ _____ events.

A sliding clamp can only be removed from the DNA if it is not _____ by any other ______. Sliding clamp loaders and DNA polymerases _______ interact with a sliding clamp at the same time because they have _______ binding sites on the sliding clamp

A sliding clamp that is bound to a DNA polymerase is not subject to ______ from the DNA. Similarly, ______ ______ factors, ______ ______ ______ proteins, and other _____ ______ proteins all interact with the same region of the sliding clamp as the clamp loader. Thus, sliding clamps are only removed from the DNA once all of the _______ that interact with them have completed their function.

At the rep fork, the _____ and ______ strands are synthesized simultaneously. This has the benefit of limiting the amount of _______ present in the cell during DNA replication.

When an ssDNA region of DNA is broken, there is a complete break in the ________ that is much more difficult to repair than an ssDNA break in a _______ region. Moreover, repair of this type of lesion frequently leads to ________ of the DNA; so limiting the time DNA is in _____ _____ form is crucial

To coordinate the replication of both DNA strands, multiple ___ _________ function at the rep fork.

In E. coli, the coordinate action of DNA polymerases is facilitated by physically linking them together in a large multiprotein complex called the ____ ______ ____ __________

________ is a general name for a multiprotein complex in which a core enzyme activity is associated with additional components that enhance function

The DNA pol III holoenzyme includes _____ copies of the core DNA pol III enzyme and one copy of the _____ subunit _____ _____ ______.

Although present in only one copy in the _______, the sliding clamp loader includes _____ copies of ____ ______

As the helicase ______ the DNA at the rep fork, the _______ strand template is exposed and acted on immediately by one ___ ____ ____ core enzyme, which synthesizes a continuous strand of complementary DNA.

The ______ strand template is not immediately acted on by DNA polymerase, instead it is _______ out as ssDNA that is rapidly bound by ______. Intermittently, ______ interacts with the DNA helicase and is activated to synthesize a new RNA primer on the ______ strand template

The resulting RNA:DNA hybrid is recognized as a _____ _____ ______ by the sliding DNA clamp loader, a sliding clamp is assmbled at this site and a second ____ _____ ____ enzyme initiates lagging strand synthesis

As one lagging strand DNA plyerase synthesizes an _______ _______, additional ssDNA is generated by the helicase and new ____ _____, is synthesized on this template.

As with the previous lagging strand primer the new RNA primer is recognized by the _____ _____ ______. Although it has traditionally been thought that there are only two _____ _____ _____ core enzymes within the the ____ _____ ____ _______, recent studies support the presence of a third

The _____ DNA pol III initiates synthesis of a new ______ _______as soon as a sliding DNA clamp is assembled on the RNA primer, likely before the completion of the previous ______ ______. Thus a second ______ ______ is thought to be initiated before the release of the polymerase synthesizing the previous _____ _____

When each Okazaki fragment is completed, the responsible DNA polymerase is ______ from the template. Because release of the DNA polymerase from the sliding clamp is a _______ process than DNA synthesis, having a second DNA polymerase dedicated to ______ strand DNA synthesis ensures the strand's continuous even during this _____ polymerase release event

Because the released DNA polymerase III core enzyme remains tethered to the ________ via the __ subunit of the sliding clamp loader, this polymerase is in an ideal position to bind the next ____ _____ _____ _______ immediately after the addition of a ______ ______. This model is called the ______ model

Why trombone model

DNA replication in euk cells also requires ______ DNA polymerases. Name them

DNA pol α/primase initiates ____ ____ while DNA pol δ & DNA pol ε _____ them. As in E. coli, one polymerase is dedicated to the ______ strand and two are dedicated to the ______ strand. Clarify which goes with which strand

What is the likely function of the several additional proteins that are known to be part of the euk. rep fork?

Interactions between rep fork proteins form the E. coli _______

The connections between the components of the DNA pol III holoenzyme aren't the only interactions at the bacterial rep fork, _____-_____ interactions between rep fork proteins facilitate rapid ______ ______ progression

replisome

The initial formation of a rep fork requires the separation of the two strands of the DNA duplex to provide the ______ necessary for ____ _______ binding and to act as a ______ for the synthesis of both the RNA primer and new DNA

Although DNA strand separation, aka _______, is most easily accomplished at _______ ____, DNA synthesis generally initiates it at _______ regions. Indeed, for ______ chromosomes, the lack of chromosome ends makes _______ DNA unwinding essential to replication initiation

Origins of replication

Replicon
**define/state an example

In contrast to E. coli, the presence of multiple origins in euk chromosomes means they have ______ replicons, one for each ______ __ _____

The replicon model proposed two components that controlled the initiation of replication, the ______ and the _______. Define both

In contrast with replicators, the origins of replication is the physical site on DNA where it is ______ and DNA synthesis ______. Although the origin is always part of the ________, sometimes in _____ cells, the origin of replication is only a _______ of the DNA sequeces required to direct the initiation of replication.

Initiator proteins have been identified in many different organisms, including ______, ______, and ______ cells. All initiator proteins select the sites that will become _____ ___ ______, although they are recruited to the DNA by ______ methods.

Initiator characteristics (4)