Lata Balakrishnan

Long Patch Base Excision Repair Proceeds via Coordinated Stimulation of the Multienzyme DNA Repair Complex

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β (pol β), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol β is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol β or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol β with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a “hit and run” mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process.

Acetylation of Dna2 Endonuclease/Helicase and Flap Endonuclease 1 by p300 Promotes DNA Stability by Creating Long Flap Intermediates

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α.

Decoding the histone H4 lysine 20 methylation mark

The molecular biology of histone H4 lysine 20 (H4K20) methylation, like many other post-translational modifications of histones, has been the subject of intensive interest in recent years. While there is an emerging consensus linking H4K20me1, H4K20me2, and H4K20me3 to transcription, repair, and constitutive heterochromatin, respectively, the specific details of these associations and the biological mechanisms by which the methylated histones are introduced and function are now the subject of active investigation. Although a large number of methylases capable of methylating H4K20 have been identified and characterized; there is no known demethylase of H4K20, though the search is ongoing. Additionally, many recent studies have been directed at understanding the role of methylated H4K20 and other histone modifications associated with different biological processes in the context of a combinatorial histone code. It seems likely that continued study of the methylation of H4K20 will yield extremely valuable insights concerning the regulation of histone modifications before and during cell division and the impact of these modifications on subsequent gene expression.

Eukaryotic Lagging Strand DNA Replication Employs A Multi-Pathway Mechanism That Protects Genome Integrity

In eukaryotic nuclear DNA replication, one strand of DNA is synthesized continuously, but the other is made as Okazaki fragments that are later joined. Discontinuous synthesis is inherently more complex, and fragmented intermediates create risks for disruptions of genome integrity. Genetic analyses and biochemical reconstitutions indicate that several parallel pathways evolved to ensure that the fragments are made and joined with integrity. An RNA primer is removed from each fragment before joining by a process involving polymerase-dependent displacement into a single-stranded flap. Evidence in vitro suggests that, with most fragments, short flaps are displaced and efficiently cleaved. Some flaps can become long, but these are also removed to allow joining. Rarely, a flap can form structure, necessitating displacement of the entire fragment. There is now evidence that post-translational protein modification regulates the flow through the pathways to favor protection of genomic information in regions of actively transcribed chromatin.

Flap Endonuclease 1 Mechanism Analysis Indicates Flap Base Binding Prior to Threading

FEN1 cleaves 5′ flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5′-end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5′-end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5′-end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.

The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1

Single-stranded DNA (ssDNA) at DNA ends is an important regulator of the DNA damage response. Resection, the generation of ssDNA, affects DNA damage checkpoint activation, DNA repair pathway choice, ssDNA-associated mutation and replication fork stability. In eukaryotes, extensive DNA resection requires the nuclease Exo1 and nuclease/helicase pair: Dna2 and Sgs1BLM. How Exo1 and Dna2-Sgs1BLM coordinate during resection remains poorly understood. The DNA damage checkpoint clamp (the 9-1-1 complex) has been reported to play an important role in stimulating resection but the exact mechanism remains unclear. Here we show that the human 9-1-1 complex enhances the cleavage of DNA by both DNA2 and EXO1 in vitro, showing that the resection-stimulatory role of the 9-1-1 complex is direct. We also show that in Saccharomyces cerevisiae, the 9-1-1 complex promotes both Dna2-Sgs1 and Exo1-dependent resection in response to uncapped telomeres. Our results suggest that the 9-1-1 complex facilitates resection by recruiting both Dna2-Sgs1 and Exo1 to sites of resection. This activity of the 9-1-1 complex in supporting resection is strongly inhibited by the checkpoint adaptor Rad953BP1. Our results provide important mechanistic insights into how DNA resection is regulated by checkpoint proteins and have implications for genome stability in eukaryotes.

Msh2-Msh3 Interferes with Okazaki Fragment Processing to Promote Trinucleotide Repeat Expansions

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntingtons disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication.

Dna2 Exhibits a Unique Strand End-dependent Helicase Function

Dna2 endonuclease/helicase participates in eukaryotic DNA transactions including cleavage of long flaps generated during Okazaki fragment processing. Its unusual substrate interaction consists of recognition and binding of the flap base, then threading over the 5′-end of the flap, and cleaving periodically to produce a terminal product ∼5 nt in length. Blocking the 5′-end prevents cleavage. The Dna2 ATP-driven 5′ to 3′ DNA helicase function promotes motion of Dna2 on the flap, presumably aiding its nuclease function. Here we demonstrate using two different nuclease-dead Dna2 mutants that on substrates simulating Okazaki fragments, Dna2 must thread onto an unblocked 5′ flap to display helicase activity. This requirement is maintained on substrates with single-stranded regions thousands of nucleotides in length. To our knowledge this is the first description of a eukaryotic helicase that cannot load onto its tracking strand internally but instead must enter from the end. Biologically, the loading requirement likely helps the helicase to coordinate with the Dna2 nuclease function to prevent creation of undesirably long flaps during DNA transactions.

Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro

Human telomeres consist of multiple tandem hexameric repeats, each containing a guanine triplet. Guanosine-rich clusters are highly susceptible to oxidative base damage, necessitating base excision repair (BER). Previous demonstration of enhanced strand displacement synthesis by the BER component DNA polymerase β in the presence of telomere protein TRF2 suggests that telomeres employ long-patch (LP) BER. Earlier analyses in vitro showed that efficiency of BER reactions is reduced in the DNA-histone environment of chromatin. Evidence presented here indicates that BER is promoted at telomeres. We found that the three proteins that contact telomere DNA, POT1, TRF1 and TRF2, enhance the rate of individual steps of LP-BER and stimulate the complete reconstituted LP-BER pathway. Thought to protect telomere DNA from degradation, these proteins still apparently evolved to allow selective access of repair proteins.

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.